Peptides that stimulate subcutaneous adipogenesis

ABSTRACT

Peptides of 5 to 14 amino acids in length that stimulate subcutaneous adipogenesis and uses thereof are provided.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional ApplicationSerial Nos. 61/778,084, filed on Mar. 12, 2013, and 61/729,626, filed onNov. 25, 2012. The entire contents of the above applications are herebyincorporated by reference.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Agreement No.LB09005060 and Contract No. DE-AC02-05CH11231 awarded by the Departmentof Energy. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to peptides for stimulatingsubcutaneous adipogenesis. The invention also relates to pharmaceuticaland cosmetic compositions containing such peptides, and to variousmethods of using such peptides and compositions to reduce scarring,improve the appearance of skin, improve tissue volume, smooth skin,recruit stem cells to formation of subcutaneous fat, reconstruct tissue,and reduce heel pain.

BACKGROUND

RHAMM is an hyaluronan (HA)-binding protein that is either poorlyexpressed or not expressed in most normal adult tissues but is highlyexpressed in aggressive human tumors (Adamia et al. (2005) Curr. DrugTargets Cardiovasc. Haematol. Disord., 5: 3-14; Tammi et al. (2002) J.Biol. Chem. 277: 4581-4584; Toole (2004) Nat. Rev. Cancer, 4: 528-539).RHAMM (gene name HMMR) is the Receptor for Hyaluronic Acid MediatedMotility, also known as CD168. RHAMM is a non-integral cell surfaceprotein (CD168) and an intracellular hyaluronan binding protein.Analyses of animal models suggest roles for RHAMM in tumorigenesis andin other disease processes such as arthritis, consistent with itswell-documented in vitro functions in cell migration and proliferationand apoptosis (Turley et al. (2002) J. Biol. Chem., 277: 4589-4592).Although cell migration and proliferation and apoptosis are essentialfunctions for morphogenesis and tissue homeostasis, genetic deletion ofRHAMM does not appear to affect embryogenesis or adult homeostasis (Tolget al. (2003) Oncogene 22: 6873-6882). To date, a primary physiologicalfunction for RHAMM has remained elusive.

RHAMM was originally isolated from subconfluent migrating fibroblasts invitro (Turley (1982) Biochem. Biophys. Res. Commun. 108: 1016-1024) andsubsequently cloned from mesenchymal cells (see, e.g., Hardwick et al.(1992) J. Cell Biol., 117: 1343-1350). Since antibodies prepared againsta shed form of RHAMM blocked HA-stimulated-fibroblast motility, RHAMMwas originally described as a cell surface protein that can transducemotogenic signaling pathways in culture (Turley et al. (2002) J. Biol.Chem. 277: 4589-4592). However, HA-bound RHAMM was later detected inintracellular compartments such as the actin and microtubulecytoskeletons, nucleus and cytoplasm (Adamia et al. (2005) Curr. DrugTargets Cardiovasc. Haematol. Disord., 5: 3-14). More recently, RHAMMhas been shown to decorate centrosomes and mitotic spindles. RHAMMappears to be required for mitotic spindle formation in culture and actson the BRCA1/BARD1 pathway to regulate mitotic spindle integrity (Joukovet al. (2006) Cancer Cell, 127: 539-52). Collectively, these resultssuggest that RHAMM may have both extracellular and intracellularfunctions, (Nickel (2005) Traffic 6: 607-614; Samuel et al. (1993) J.Cell Biol., 123: 749-758; Zhang et al. (1998) J. Biol. Chem., 273:11342-11348) thus resembling a group of proteins includingepimorphin/syntaxin-2, and autocrine motility factor/phosphoglucoseisomerase that are also found at the cell surface where they transmitsignals across the cell membrane even though, like RHAMM, they lack bothGolgi-ER export peptides and membrane spanning sequences (Nickel (2005)Traffic 6: 607-614).

Although the intracellular versus extracellular functions of RHAMM havenot yet been clearly dissected, accumulating data suggest that bothforms may contribute to mesenchymal phenotypes, at least during disease.For example, RHAMM expression in culture is increased in transformedfibroblasts by fibrogenic cytokines such as TGF-β (Samuel et al. (1993)J. Cell Biol., 123: 749-758). Cell surface RHAMM is required foractivation through fibrogenic cytokines such as PDGF (Zhang et al.(1998) J. Biol. Chem., 273: 11342-11348). It has also been demonstratedthat RHAMM expression is high in clinically aggressive mesenchymaltumors (fibromatoses or desmoid tumors) (see, e.g., Tolg et al. (2003)Oncogene 22: 6873-6882). In a mouse model susceptible to desmoid andupper intestinal tract tumors, genetic deletion of RHAMM stronglyreduces desmoid initiation and invasion but not upper intestinal tracttumors. Fibroproliferative processes such as aggressive fibromatosisresemble proliferative/migratory stages of wound healing (Cheon et al.(2002) Proc. Natl. Acad. Sci. USA, 99: 6973-6978). Furthermore, theexpression of RHAMM is modulated during wounding (Lovvorn et al. (1998)J. Pediatr. Surg., 33: 1062-1069; discussion 1069-1070).

It has been found that factors that regulate fibroblast function playdual roles in wound repair and tumorigenesis (Bissell (2001) Exp. Mol.Med., 33: 179-190; Park et al. (2000) Mol. Med. Today, 6: 324-329) andmesenchymal stem cell trafficking/differentiation into wound sites hasbecome a topic of study (see, e.g., Fu et al. (2006) Wound RepairRegen., 14: 325-35; Mansilla et al. (2006) Transplant Proc. 38: 967-969;Shumakov (2003) Bull. Exp. Biol. Med., 136: 192-195). Mesenchymal stemcells and resident fibroblasts in wounds have immune-modulatoryfunctions that affect the timing and extent of fibrosis during woundrepair (Domaszewska and Oszewski (2006) Ann. Transplnt. 11: 45-52).

Genetic loss of RHAMM or blocking RHAMM function using peptidesmimicking its hyaluronan binding sequence or antibodies to this sequencehave been shown to promote subcutaneous adipogenesis. One such peptide(STMMSRSHKTRSHHV (SEQ ID NO: 1), P-1 peptide, also referred to herein aspeptide P15-1) isolated from a random phage library has been shown tobind to hyaluronan, is adipogenic and resembles the hyaluronan bindingregion of RHAMM, a mesenchymal factor involved in wound repair. Anotherpeptide, peptide B (KLKDENSQLKSEVSK (SEQ ID NO: 2)), which containsseveral key residues required for an interaction of RHAMM with HA, isstrongly adipogenic. It was reported in PCT Publication No. WO2008/140586 that RHAMM displays an effect in modulation of adiposetissue development. In particular, histology analysis of tissue sectionsthrough unwounded skin of RHAMM −/− mice showed that the subcutaneouslayer of fat was two to three times thicker than in wild-type littermateskin and fibroblasts grown from RHAMM −/− wounds incorporated highlevels of fat droplets and reduced smooth muscle actin. Furthermore,RHAMM −/− dermal fibroblasts converted to adipocytes when grown inadipogenic medium. In contrast fibroblasts grown from litter matchedwild-type wounds did not exhibit fat droplets, and expressed abundantsmooth muscle actin. RHAMM-rescued dermal fibroblasts do not undergoadipogenic conversion when grown in adipogenic medium. Conversely, imageanalysis of RHAMM −/− mice showed that they have significantly lessvisceral fat and a lower bone density than wild-type litter mates. Thesedata were said to indicate that RHAMM has a differential effect onvisceral vs. subcutaneous adipogenesis.

It is also believed that RHAMM is selective in its regulation ofsubcutaneous vs. visceral fat, and that this regulation is associatedwith effects on bone marrow stem cells since bone density provides ameasure of stem cell activity. This indicates that RHAMM affectssubcutaneous fat deposition through its ability to regulate mesenchymalstem cell differentiation, a conclusion substantiated by the effect ofRHAMM loss on another mesenchymal stem cell type, myofibroblasts.Furthermore, hyaluronan/RHAMM interactions play a role in this effect onmesenchymal differentiation since HA binding peptides also promoteadipogenesis. In addition to these in vivo effects, RHAMM −/− dermalfibroblasts spontaneously develop into adipocytes when cultures becomecrowded while wild type dermal fibroblasts do not.

SUMMARY

An aspect of the present invention is directed to a peptide forstimulating subcutaneous adipogenesis, wherein the peptide has a lengthof 6 to 14 amino acids and comprises a motif having the sequence:X¹-X²-X³-X⁴-X⁵-X⁶ wherein at least one of X¹ and X⁶ is a positivelycharged amino acid; the other of X¹ and X⁶ is a positively charged aminoacid, leucine (L), or alanine (A); X² is serine (S), glutamine (Q),alanine (A), glutamate (E), asparagine (N), proline (P), cysteine (C),lysine (K), aspartate (D), tryptophan (W), methionine (M), or threonine(T); X³ is a negatively charged amino acid, alanine (A), glutamine (Q),serine (S), lysine (K), asparagine (N), or threonine (T); X⁴ isisoleucine (I), valine (V), leucine (L), asparagine (N), alanine (A),serine (S), proline (P), threonine (T), or glutamine (Q); and X⁵ isserine (S), aspartate (D), threonine (T), leucine (L), alanine (A),phenylalanine (F), glutamate (E), asparagine (N), glycine (G), arginine(R), glutamine (Q), histidine (H), or isoleucine (I); provided that X²and X⁵ are not both glutamine (Q) and provided that when X¹ is arginine(R), X³ is not lysine (K). Preferably, the peptide is KSEVSK (SEQ ID NO:3), KQEVSK (SEQ ID NO: 4), KQEVDK (SEQ ID NO: 5), KQENTK (SEQ ID NO: 6),KSEVLK (SEQ ID NO: 7), KQDVSK (SEQ ID NO: 8), KQELDR (SEQ ID NO: 9),LEEIFK (SEQ ID NO: 10), LSELEK (SEQ ID NO: 11), KSEISK (SEQ ID NO: 12),KNEVSK (SEQ ID NO: 13), KSEVTK (SEQ ID NO: 14), KSEVNK (SEQ ID NO: 15),KSDVSK (SEQ ID NO: 16), KSQVSK (SEQ ID NO: 17), KPEVSK (SEQ ID NO: 18),KSEVGK (SEQ ID NO: 19), KSDSSK (SEQ ID NO: 20), KSSPSK (SEQ ID NO: 21),KSEASK (SEQ ID NO: 22), KSELRK (SEQ ID NO:23), KCEVSK (SEQ ID NO: 24),KSKPSK (SEQ ID NO: 25), KKEVSK (SEQ ID NO: 26), KEEVSK (SEQ ID NO: 27),KSETSK (SEQ ID NO: 28), KSNVSK (SEQ ID NO: 29), KDEVSK (SEQ ID NO: 30),KSEVEK (SEQ ID NO: 31), KSAVSK (SEQ ID NO: 32), KWEVSK (SEQ ID NO: 33),KMEVSK (SEQ ID NO: 34), KSEVQK (SEQ ID NO: 35), KSEVHK (SEQ ID NO: 36),KSSVSK (SEQ ID NO: 37), ASEVSK (SEQ ID NO: 38), KAEVSK (SEQ ID NO: 39),KSEVAK (SEQ ID NO: 40), KSEVSA (SEQ ID NO: 41), LKSEVSK (SEQ ID NO: 42),QLKSEVSK (SEQ ID NO: 43), SQLKSEVSK (SEQ ID NO: 44), NSQLKSEVSK (SEQ IDNO: 45), ENSQLKSEVSK (SEQ ID NO: 46), DENSQLKSEVSK (SEQ ID NO: 47),KDENSQLKSEVSK (SEQ ID NO: 48), or LKDENSQLKSEVSK (SEQ ID NO: 49).

Another aspect of the invention is directed to a peptide for stimulatingsubcutaneous adipogenesis, wherein the peptide has a length of 5 to 14amino acids and comprises a motif having the sequence: X¹-X²-X³-X⁴-X⁵-X⁶wherein at least one of X¹ and X⁶ is a positively charged amino acid;the other of X¹ and X⁶ is a positively charged amino acid, leucine (L)or a conservative substitution therefor, alanine (A) or a conservativesubstitution therefor, or absent; and X²-X³-X⁴-X⁵ is an amino acidsequence having at least 50% sequence identity with the amino acidsequence SEVS (SEQ ID NO: 50) and having a length of 4 to 8 amino acids.

Yet another aspect of the invention is directed to a peptide forstimulating subcutaneous adipogenesis, wherein the peptide has a lengthof 6 to 13 amino acids and comprises a motif having the sequence:X¹—X²-X³-X⁴-X⁵-X⁶ wherein X¹ is serine (S) or a conservativesubstitution therefor; X² is a hydrophobic amino acid; X³ is any aminoacid; X⁴ is a hydrophobic amino acid; X⁵ is serine (S), arginine (R), ora conservative substitution therefor; and X⁶ is a charged amino acid;provided that X¹, X² and X⁴ are not all glycine (G) and provided that X¹is not glutamine (Q).

Preferably, the peptide is STMMSR (SEQ ID NO: 51), SIMMSR (SEQ ID NO:52), STLMSR (SEQ ID NO: 53), STVMSR (SEQ ID NO: 54), STGLSR (SEQ ID NO:55), STTMSR (SEQ ID NO: 56), STRMSR (SEQ ID NO: 57), STLMRR (SEQ ID NO:58), STPVSR (SEQ ID NO: 59), STMMSRS (SEQ ID NO: 60), STMMSRSH (SEQ IDNO: 61), STMMSRSHK (SEQ ID NO: 62), STMMSRSHKT (SEQ ID NO: 63),STMMSRSHKTR (SEQ ID NO: 64), STMMSRSHKTRS (SEQ ID NO: 65), orSTMMSRSHKTRSH (SEQ ID NO: 66).

Another aspect of the present invention is directed to a peptide forstimulating subcutaneous adipogenesis, wherein the peptide has a lengthof 6 to 13 amino acids and comprises a motif having the sequence:X¹-X²-X³-X⁴-X⁵-X⁶-X⁵-X⁶ wherein X¹ is serine (S) or a conservativesubstitution therefor; X⁶ is a charged amino acid; and X²-X³-X⁴-X⁵ is anamino acid sequence having at least 50% sequence identity with the aminoacid sequence TMMS (SEQ ID NO: 67) and having a length of 2 to 8 aminoacids.

A further aspect of the invention is directed to a pharmaceutical orcosmetic composition comprising any one or more of the above peptidesand a pharmaceutically or cosmetically acceptable carrier.

Yet another aspect of the invention is directed to a method of reducingscarring comprising administering any one or more of the above peptidesor the composition to a subject in an amount sufficient to reduce thearea of scarring or improve the appearance of a scarred area.

An aspect of the invention is directed to a method of improving theappearance of skin comprising administering any one or more of the abovepeptides or the composition to a subject in an amount sufficient toimprove the appearance of skin in an area of a subject.

Another aspect of the invention is directed to a method of improvingtissue volume in an area of a subject, said method comprisingadministering any one or more of the above peptides or the compositionto the subject in an amount sufficient to increase the tissue volume inthe area.

Yet another aspect of the invention is directed to a method of smoothingskin in an area of a subject comprising administering any one or more ofthe above peptides or the composition to the subject in an amountsufficient to smooth skin in the area.

A further aspect of the invention is directed to a method of recruitingstem cells to formation of subcutaneous fat in a subject, the methodcomprising administering any one or more of the above peptides or thecomposition to the subject in an amount sufficient to recruit stem cellsto the formation of subcutaneous fat in the subject.

Another aspect of the invention is directed to a method ofreconstructing tissue of a subject comprising administering any one ormore of the above peptides or the composition to the tissue of thesubject in an amount sufficient to increase the volume of the tissueduring or after a tissue reconstruction procedure.

Yet another aspect of the invention is directed to a method of reducingheel pain in a subject comprising administering any one or more of theabove peptides or the composition to the subject in an amount sufficientto reduce heel pain in the subject during walking.

Definitions

The term “peptide” as used herein refers to a polymer of amino acidresidues typically ranging in length from about 5 to about 14 aminoacids. The amino acid residues of the peptide can be “L-form” amino acidresidues, “D” amino acid residues, or a combination thereof. L-, D-, orβ-amino acid versions of the peptide sequence as well as retro, inverso,and retro-inverso isoforms are included. “β-peptides” are comprised of“β amino acids”, which have their amino group bonded to the β carbonrather than the α-carbon as in the 20 standard biological amino acids.

The terms “standard” and “natural” as applied to peptides herein referto peptides constructed only from the standard naturally-occurring aminoacids: alanine (Ala, A), cysteine (Cys, C), aspartate (Asp, D),glutamate (Glu, E), phenylalanine (Phe, F), glycine (Gly, G), histidine(His, H), isoleucine (Ile, I), lysine (Lys, K), leucine (Leu, L),methionine (Met, M), asparagine (Asn, N), proline (Pro, P), glutamine(Gln, Q), arginine (Arg, R), serine (Ser, S), threonine (Thr, T), valine(Val, V), tryptophan (Trp, W), and tyrosine (Tyr, Y). A peptide of theinvention “corresponds” to a natural peptide if it elicits a biologicalactivity (e.g., adipogenic activity) related to the biological activityand/or specificity of the naturally occurring peptide. The elicitedactivity may be the same as, greater than or less than that of thenatural peptide.

The terms “non-standard,” and “non-natural,” and “analogue” as appliedto peptides herein refer to peptides which contain one or more aminoacids which are not standard amino acids and/or which are notnaturally-occurring amino acids. A skilled artisan would be familiarsuch non-standard and non-naturally occurring amino acids and would beable to select suitable non-standard and non-naturally occurring aminoacids for use in the peptides of the present invention. Amino acidanalogues include amino acids that occur in nature but which arenon-standard (e.g., norvaline, norleucine), as well as synthetic aminoacids that do not occur in nature. For example, various amino acidanalogues include, but are not limited to 2-aminoadipic acid,3-aminoadipic acid, beta-alanine (beta-aminopropionic acid),2-aminobutyric acid, 4-aminobutyric acid, piperidinic acid,6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid,3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminobutyric acid,desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid,n-ethylglycine, n-ethylasparagine, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,n-methylglycine, sarcosine, n-methylisoleucine, 6-n-methyllysine,n-methylvaline, norvaline, norleucine, ornithine, and the like. Thesemodified amino acids are illustrative and not intended to be limiting.

The term “conservative substitution” is used to refer to an amino acidsubstitution that does not substantially alter the activity (e.g.,adipogenic activity and/or specificity) of the peptide. Conventionalconservative amino acid substitutions involve substituting one aminoacid for another amino acid with similar structure and chemicalproperties (e.g., charge or hydrophobicity). Such conventionalsubstitutions include, but are not limited to, the following: 1) glycine(G)/alanine (A), 2) arginine (R)/lysine (K), 3) serine (S)/threonine(T)/tyrosine (Y), 4) leucine (L)/isoleucine (I)/valine (V), 5) asparticacid (D)/glutamic acid (E), 6) glutamine (Q)/asparagine (N), and 7)phenylalanine (F)/tyrosine (Y)/tryptophan (W). Other functionalconservative substitutions include, but are not limited to 8) glycine(G)/alanine (A)/proline (P), 9) tyrosine (Y)/histidine (H), 10) arginine(R)/lysine (K)/histidine (H), 11) serine (S)/threonine (T)/cysteine (C),12) leucine (L)/isoleucine (I)/valine (V)/methionine (M), 13) alanine(A)/serine (S)/threonine (T)/methionine (M)/glycine (G), and 14)methionine (M)/lysine (K) (under hydrophobic conditions). Conservativesubstitutions also include “analog substitutions” where a standard aminoacid is replaced by a non-standard (e.g., rare, synthetic, etc.) aminoacid differing minimally from the standard amino acid. Such analogsubstitutions can be derived synthetically from the standard aminoacids, are isomers, or are metabolite precursors. Examples of such“analog substitutions” include, but are not limited to, 1) Lys→ornithine(Orn), 2) Leu→Norleucine, 3) Lys→Lys[TFA], 4) Phe→phenylglycine, and 5)δ-amino butylglycine→δ-amino hexylglycine, where [TFA] refers totrifluoroacetyl. Conservative substitutions also include a replacementof an amino acid with another amino acid having shared properties asshown in FIG. 1. Such substitutions include substitutions of one aminoacid in one of the following groups with another amino acid in the samegroup: 1) aromatic amino acids: phenylalanine (F)/tyrosine (Y)/histidine(H)/tryptophan (W); 2) aliphatic amino acids: isoleucine (I)/valine(V)/leucine(L)/alanine (A)/glycine (G); 3) positively charged aminoacids: histidine (H)/lysine (K)/arginine (R); 4) negatively chargedamino acids: aspartate (D)/glutamate (E); and 5) tiny amino acids:cysteine (C)/alanine (A)/glycine (G)/serine (S). Conservativesubstitutions also include substitutions of amino acids having unchargedpolar side chains, i.e., serine (S)/threonine (T)/asparagine(N)/glutamine (Q)/tyrosine (Y)/cysteine(S). Where amino acid sequencesare disclosed herein, amino acid sequences comprising one or more of theabove-identified conservative substitutions are also contemplated.

The term percent “identity” refers to two or more sequences that have aspecified percentage of amino acid residues that are the same, whencompared and aligned for maximum correspondence, as measured usingstandard sequence comparison algorithms or by visual inspection. Forsequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. By a peptide having anamino acid sequence with at least, for example, 50% identity to areference amino acid sequence, it is intended that up to 50% of theamino acid residues in the test sequence may be inserted, deleted, orsubstituted with another amino acid. These alterations of the testsequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence. Sequence identity of a test sequence is determinedover the full length of the reference peptide or over a specifiedportion of the reference peptide. For example, in considering the aminoacid sequence TMMS (SEQ ID NO: 67) as the reference sequence of the fullsequence STMMSR (SEQ ID NO: 51), some test sequences with amino acidsubstitutions (e.g., TMAQ (SEQ ID NO: 68), TRMW (SEQ ID NO: 69), TVLS(SEQ ID NO: 70), IGMS (SEQ ID NO: 71), and KMTS (SEQ ID NO: 72)) have50% sequence identity with the reference sequence, as do some testsequences with amino acid deletions (e.g., MM, MS, TM, TS), and sometest sequences with amino acid insertions (e.g., TMMAAAAS (SEQ ID NO:73), TAAMMSAA(SEQ ID NO: 74), TMAAAMAS (SEQ ID NO: 75)). Some testsequences with insertions, deletions, and/or substitutions will alsohave 50% sequence identity with the reference sequence. For example,again considering the amino acid sequence TMMS (SEQ ID NO: 67) as thereference sequence, sequences such as TCS or AMS (having one deletionand one substitution as compared to the reference sequence), or TLLMMT(SEQ ID NO: 76) or AMLIMS (SEQ ID NO: 77) (having two insertions and onesubstitution as compared to the reference sequence), have 50% identitywith the reference sequence, as can be seen from the following sequencealignments:

TMMS T-CS TMMS -AMS T--MMS TLLMMT TM--MS AMLIMSBy contrast, sequences such as TAAM (SEQ ID NO: 78) or FTMA (SEQ ID NO:79) (having one substitution, one insertion, and one deletion ascompared to the reference sequence), TCCRS (SEQ ID NO: 80) (having oneinsertion and two substitutions as compared to the reference sequence),or TLALM (SEQ ID NO: 81) (having two insertions, one substitution, andone deletion as compared to the reference sequence) have less than 50%sequence identity with the reference sequence TMMS (SEQ ID NO: 67), ascan be seen in the following sequence alignments:

TM-MS TAAM- (40% sequence identity) -TMMS FTMA- (40% sequence identity)TMM-S TCCRS (40% sequence identity) T-M-MS TLALM-(33% sequence identity)

When using a sequence comparison algorithm, test and reference sequencesare entered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated. Thesequence comparison algorithm then calculates the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated program parameters. Optimal alignment ofsequences for comparison can be conducted, for example, by the localhomology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2: 482,by the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol.Biol. 48: 443, by the search for similarity method of Pearson & Lipman(1988) Proc. Natl. Acad. Sci., USA, 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection.

One illustrative but non-limiting method for determining a globalsequence alignment is through use of the FASTDB computer program basedon the algorithm of Brutlag et al. (1990) Comp. App. Biosci. 6: 237-245.Use of such alignment algorithms is known to one of ordinary skill inthe art. If the test sequence is shorter than the reference sequence dueto N- or C-terminal deletions, not because of internal deletions, amanual correction is typically made to the results. This is because theFASTDB program does not account for N- and C-terminal truncations of thetest sequence when calculating global percent identity. For testsequences truncated at the N- and C-termini, relative to the referencesequence, the percent identity is corrected by calculating the number ofresidues of the reference sequence that are N- and C-terminal of thetest sequence, which are not matched/aligned with a corresponding testresidue, as a percent of the total bases of the reference sequence.Whether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thepresent invention. Only residues to the N- and C-termini of the testsequence, that are not matched/aligned with the reference sequence, aretypically considered for the purposes of manually adjusting the percentidentity score. That is, only reference residue positions outside thefarthest N- and C-terminal residues of the test sequence. For example, a3-amino acid residue test sequence is aligned with a 4-residue referencesequence to determine percent identity. The deletion occurs at theN-terminus of the test sequence and therefore, the FASTDB alignment doesnot show a matching/alignment of the first residue at the N-terminus.The unpaired residue represents 25% of the sequence (number of residuesat the N and C-termini not matched/total number of residues in thereference sequence) so 25% is subtracted from the percent identity scorecalculated by the FASTDB program. If the remaining 3 residues wereperfectly matched the final percent identity would be 75%. In anotherexample, a 3 residue test sequence is compared with a 4 residuereference sequence. This time the deletions are internal deletions sothere are no residues at the N- or C-termini of the test sequence whichare not matched/aligned with the reference. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the testsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the reference sequence, are manually corrected for.No other manual corrections are typically made or necessary.

The term “variant” refers to a peptide differing from a peptide of theinvention, but retaining essential properties thereof (e.g., the abilityto inhibit RHAMM activity). Generally, variants are overall closelysimilar, and may be identical, in many regions, to a peptide sequence ofthe invention. Besides conservative amino acid substitution, a variantpeptide can include (i) substitutions with one or more of non-conservedamino acid residues, where the substituted amino acid residues may ormay not be a standard amino acid and may or may not be a naturallyoccurring amino acid, or (ii) conservative substitutions with one ormore non-standard or non-naturally occurring amino acid residues, or(iii) fusion of the peptide with another compound, such as a compound toincrease the stability and/or solubility of the peptide (for example,polyethylene glycol), or (iv) fusion of the peptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Methods of making such variant peptides are within the skill of one ofordinary skill in the art. For example, peptide variants containingamino acid substitutions of charged amino acids with other charged orneutral amino acids may produce peptides with improved characteristics,such as less aggregation. Aggregation of pharmaceutical formulationsboth reduces activity and increases clearance due to the aggregate'simmunogenic activity (see, e.g., Pinckard et al. (1967) Clin. Exp.Immunol. 2: 331340; Robbins et al. (1987) Diabetes 36: 838-845; Clelandet al. (1993) Crit. Rev. Therapeutic Drug Carrier Systems, 10: 307-377).

The terms “subject,” “individual,” and “patient” may be usedinterchangeably and refer to a mammal, preferably a human or a non-humanprimate, but also domesticated mammals (e.g., canine or feline),laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig) andagricultural mammals (e.g., equine, bovine, porcine, ovine). The subjectcan be a human (e.g., adult male, adult female, adolescent male,adolescent female, male child, female child) under the care of aphysician or other health worker in a hospital, as an outpatient, orother clinical context. Alternatively, the subject may not be under thecare or prescription of a physician or other health worker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the classification used herein for variousnaturally-occurring standard amino acids. It will be recognized thatnon-standard and/or non-naturally occurring amino acids can also fallinto this classification.

FIG. 2 illustrates the RHAMM HA binding region and the adipogenicsubdomain.

FIGS. 3A-3C provide illustrative adipogenesis dose-response data forpeptide B (SEQ ID NO: 2).

FIG. 4 shows illustrative micrographs showing lipid accumulation incells in response to treatment with peptide B (SEQ ID NO: 2).

FIGS. 5A-5B show illustrative results from experiments using peptide B(SEQ ID NO: 2; 25-50 μg/injection site) or an adipogenic antibody in arat dermal skin model of adipogenesis.

FIG. 6 shows illustrative results of testing of peptide B (SEQ ID NO: 2)in nude guinea pigs. An increase in subcutaneous fat deposition wasobserved on the upper back of these animals and in the ear fat pad afterinjection of peptide B in both locations. Arrows point to subcutaneousfat layer.

FIG. 7 provides illustrative data showing the adipogenic activity ofvarious peptides.

FIGS. 8A and 8B show illustrative dose-response adipogenesis data forthe 6-mer peptide KSEVSK (SEQ ID NO: 3).

FIG. 9 provides illustrative micrographs showing lipid accumulation incells in response to treatment with the 9-, 7-, and 6-mers (SEQ ID NOs:44, 42, and 3, respectively).

FIG. 10 shows illustrative results of in vivo testing of the 6-merKSEVSK (SEQ ID NO: 3) for an adipogenic effect.

FIGS. 11 and 12 show illustrative adipogenic activity data for peptidesin which flanking lysine residues have been mutated to alanine residues(ASEVSK (SEQ ID NO: 38) and KSEVSA (SEQ ID NO: 41)).

FIGS. 13A and 13B illustrate the adipogenic activity of two five-merpeptides (KSEVS (SEQ ID NO. 82), FIG. 13A) and (SEVSK (SEQ ID NO: 83),FIG. 13B).

DETAILED DESCRIPTION

Peptides and mimetics are identified that promote subcutaneousadipogenesis without inducing undesired visceral adipogenesis. Thesepeptides are useful in a variety of cosmetic and therapeuticapplications. Without being bound to a particular theory, it is believedthat the peptides described herein achieve the adipogenic activity byinhibiting (fully or partially) RHAMM binding to hyaluronan and/or RHAMMbiological activity (e.g., binding to CD44). The peptides of theinvention are short in length having only about 5 to 14 amino acids ascompared to other peptides known in the art to have adipogenic activity,such as the P-1 Peptide (peptide P15-1; SEQ ID NO: 1). The “short”peptides offer certain advantages such as reduced cost of production,reduced immunogenicity, increased activity, and increased penetrationinto skin when applied topically.

Peptide P15-1 (SEQ ID NO: 1) was isolated by screening a random phagelibrary for peptides having the ability to bind to hyaluronan (HA).Alignments were performed to assess the similarity of the P15-1 (SEQ IDNO: 1) peptide to peptide B (SEQ ID NO: 2), a peptide derived from theHA-binding region of RHAMM. Since these peptides bind to hyaluronan andresemble a RHAMM sequence and have been shown to be adipogenic, it waspossible to identify conserved motifs that are believed to beresponsible for the adipogenic properties of the peptides.

It is also believed that the use of RHAMM-inhibiting peptides describedherein provides signals that force differentiation into adipocytes. Inaddition, RHAMM −/− dermal fibroblasts express very low levels of smoothmuscle actin indicating that RHAMM also regulates development of thismesenchymal stem cell lineage.

With respect to mesenchymal and other skin stem cells, a uniqueadvantage of using the RHAMM-inhibiting peptides described herein forincreasing subcutaneous fat is that its effects are selective. Thus,visceral fat, whose increased accumulation on body organs is associatedwith disease is decreased while subcutaneous fat is increased. This isan unusual effect and differentiates RHAMM's effects from otheradipocyte promoting factors such as leptin that affect accumulation ofboth types of adipocytes. Accordingly, it is believed the peptidesdescribed herein will not increase visceral fat.

Another property of RHAMM is related to its very restricted expressionin the adult human tissues. RHAMM is poorly expressed or not expressedphysiologically but is increased following tissue injury ortransformation to the neoplastic state. Therefore, the RHAMM-inhibitorypeptides described herein should have low toxicity. In fact, it isbelieved that blocking RHAMM function can be beneficial to those with atumor load or with inflammation-based diseases such as arthritis sinceRHAMM has pro-oncogenic, pro-inflammatory functions.

RHAMM Inhibiting Peptides.

Peptides are provided that inhibit RHAMM biological activity and therebyinduce or stimulate subcutaneous adipogenesis. In one aspect of theinvention, the peptide that stimulates subcutaneous adipogenesis has alength of 6 to 14 amino acids and comprises a motif having the sequence:

-   -   X¹-X²-X³-X⁴-X⁵-X⁶        wherein at least one of X¹ and X⁶ is a positively charged amino        acid and the other of X¹ and X⁶ is a positively charged amino        acid, leucine (L), or alanine (A). X² is serine (S), glutamine        (Q), alanine (A), glutamate (E), asparagine (N), proline (P),        cysteine (C), lysine (K), aspartate (D), tryptophan (W),        methionine (M), or threonine (T). X³ is a negatively charged        amino acid, alanine (A), glutamine (Q), serine (S), lysine (K),        asparagine (N), or threonine (T). X⁴ is isoleucine (I), valine        (V), leucine (L), asparagine (N), alanine (A), serine (S),        proline (P), threonine (T), or glutamine (Q); and X⁵ is serine        (S), aspartate (D), threonine (T), leucine (L), alanine (A),        phenylalanine (F), glutamate (E), asparagine (N), glycine (G),        arginine (R), glutamine (Q), histidine (H), or isoleucine (I);        provided that X² and X⁵ are not both glutamine (Q) and provided        that when X¹ is arginine (R), X³ is not lysine (K).

The length of the peptide can be, for example, 13 amino acids, 12 aminoacids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7amino acids, or 6 amino acids.

In certain aspects of the invention, both X¹ and X⁶ are positivelycharged amino acids.

X¹ is preferably lysine (K).

X⁶ is preferably lysine (K) or arginine (R), and is more preferablylysine (K).

In some aspects of the invention, X² is serine (S), glutamine (Q),alanine (A), glutamate (E), asparagine (N), proline (P), cysteine (C),lysine (K), aspartate (D), tryptophan (W), or methionine (M). X² ispreferably serine (S) or glutamine (Q). More preferably, X² is serine(S).

X³ can be a negatively charged amino acid, alanine (A), glutamine (Q),serine (S), lysine (K), or asparagine (N). For example, X³ can be anegatively charged amino acid (e.g., glutamate (E), aspartate (D)). X³is preferably glutamate (E).

X⁴ can be isoleucine (I), valine (V), leucine (L), asparagine (N),alanine (A), serine (S), proline (P), or threonine (T). For example, X⁴can be isoleucine (I), valine (V), or leucine (L). X⁴ is preferablyvaline.

X⁵ can be serine (S), aspartate (D), threonine (T), leucine (L), alanine(A), phenylalanine (F), glutamate (E), asparagine (N), glycine (G),arginine (R), glutamine (Q), or histidine (H). For example, X⁵ can beserine (S), aspartate (D), threonine (T), or glutamate (E). X⁵ ispreferably serine (S).

For example, in certain peptides of the present invention, X¹ is lysine(K), X² is serine (S), X³ is glutamate (E), X⁴ is valine (V), X⁵ isserine (S), and X⁶ is lysine (K).

In certain peptides of the invention, the motif is KSEVSK (SEQ ID NO:3), KQEVSK (SEQ ID NO: 4), KQEVDK (SEQ ID NO: 5), KQENTK (SEQ ID NO: 6),KSEVLK (SEQ ID NO: 7), KQDVSK (SEQ ID NO: 8), KQELDR (SEQ ID NO: 9),LEEIFK (SEQ ID NO: 10), LSELEK (SEQ ID NO: 11), KSEISK (SEQ ID NO: 12),KNEVSK (SEQ ID NO: 13), KSEVTK (SEQ ID NO: 14), KSEVNK (SEQ ID NO: 15),KSDVSK (SEQ ID NO: 16), KSQVSK (SEQ ID NO: 17), KPEVSK (SEQ ID NO: 18),KSEVGK (SEQ ID NO: 19), KSDSSK (SEQ ID NO: 20), KSSPSK (SEQ ID NO: 21),KSEASK (SEQ ID NO: 22), KSELRK (SEQ ID NO:23), KCEVSK (SEQ ID NO: 24),KSKPSK (SEQ ID NO: 25), KKEVSK (SEQ ID NO: 26), KEEVSK (SEQ ID NO: 27),KSETSK (SEQ ID NO: 28), KSNVSK (SEQ ID NO: 29), KDEVSK (SEQ ID NO: 30),KSEVEK (SEQ ID NO: 31), KSAVSK (SEQ ID NO: 32), KWEVSK (SEQ ID NO: 33),KMEVSK (SEQ ID NO: 34), KSEVQK (SEQ ID NO: 35), KSEVHK (SEQ ID NO: 36),KSSVSK (SEQ ID NO: 37), ASEVSK (SEQ ID NO: 38), KAEVSK (SEQ ID NO: 39),KSEVAK (SEQ ID NO: 40), or KSEVSA (SEQ ID NO: 41). For example, themotif can be KSEVSK (SEQ ID NO: 3).

In some aspects of the invention, the peptide for stimulatingsubcutaneous adipogenesis is KSEVSK (SEQ ID NO: 3), KQEVSK (SEQ ID NO:4), KQEVDK (SEQ ID NO: 5), KQENTK (SEQ ID NO: 6), KSEVLK (SEQ ID NO: 7),KQDVSK (SEQ ID NO: 8), KQELDR (SEQ ID NO: 9), LEEIFK (SEQ ID NO: 10),LSELEK (SEQ ID NO: 11), KSEISK (SEQ ID NO: 12), KNEVSK (SEQ ID NO: 13),KSEVTK (SEQ ID NO: 14), KSEVNK (SEQ ID NO: 15), KSDVSK (SEQ ID NO: 16),KSQVSK (SEQ ID NO: 17), KPEVSK (SEQ ID NO: 18), KSEVGK (SEQ ID NO: 19),KSDSSK (SEQ ID NO: 20), KSSPSK (SEQ ID NO: 21), KSEASK (SEQ ID NO: 22),KSELRK (SEQ ID NO:23), KCEVSK (SEQ ID NO: 24), KSKPSK (SEQ ID NO: 25),KKEVSK (SEQ ID NO: 26), KEEVSK (SEQ ID NO: 27), KSETSK (SEQ ID NO: 28),KSNVSK (SEQ ID NO: 29), KDEVSK (SEQ ID NO: 30), KSEVEK (SEQ ID NO: 31),KSAVSK (SEQ ID NO: 32), KWEVSK (SEQ ID NO: 33), KMEVSK (SEQ ID NO: 34),KSEVQK (SEQ ID NO: 35), KSEVHK (SEQ ID NO: 36), KSSVSK (SEQ ID NO: 37),ASEVSK (SEQ ID NO: 38), KAEVSK (SEQ ID NO: 39), KSEVAK (SEQ ID NO: 40),KSEVSA (SEQ ID NO: 41), LKSEVSK (SEQ ID NO: 42), QLKSEVSK (SEQ ID NO:43), SQLKSEVSK (SEQ ID NO: 44), NSQLKSEVSK (SEQ ID NO: 45), ENSQLKSEVSK(SEQ ID NO: 46), DENSQLKSEVSK (SEQ ID NO: 47), KDENSQLKSEVSK (SEQ ID NO:48), or LKDENSQLKSEVSK (SEQ ID NO: 49). For example, the peptide can beKSEVSK (SEQ ID NO: 3), LKSEVSK (SEQ ID NO: 42), QLKSEVSK (SEQ ID NO:43), or SQLKSEVSK (SEQ ID NO: 44). Preferred peptides include KSEVSK(SEQ ID NO: 3), LKSEVSK (SEQ ID NO: 42), or SQLKSEVSK (SEQ ID NO: 44).KSEVSK (SEQ ID NO: 3) is a more preferred peptide.

In certain aspects of the invention, the motif is not RQKVLK (SEQ ID NO:84) or LQATQK (SEQ ID NO: 85).

Another aspect of the present invention relates to peptides forstimulating subcutaneous adipogenesis. The peptide has a length of 5 to14 amino acids and comprises a motif having the sequence

-   -   X¹-X²-X³-X⁴-X⁵-X⁶        wherein at least one of X¹ and X⁶ is a positively charged amino        acid, and the other of X¹ and X⁶ is a positively charged amino        acid, leucine (L) or a conservative substitution therefor,        alanine (A) or a conservative substitution therefor, or absent.        In such peptides, X²—X³-X⁴-X⁵ is an amino acid sequence having        at least 50% sequence identity with the amino acid sequence SEVS        (SEQ ID NO: 50) and having a length of 4 to 8 amino acids.

The length of the peptide can be 6 to 14 amino acids. For example, thelength of the peptide can be 13 amino acids, 12 amino acids, 11 aminoacids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, or 6amino acids.

In some aspects of the invention, the length of X²-X³-X⁴-X⁵ is 4 to 6amino acids, or 4 amino acids.

X²-X³-X⁴-X⁵ can have at least about 75% sequence identity with the aminoacid sequence SEVS (SEQ ID NO: 50).

In certain aspects, both X¹ and X⁶ are positively charged amino acids.

X¹ is preferably lysine (K).

X⁶ is preferably lysine (K) or arginine (R), and is more preferablylysine (K).

Yet another aspect of the present invention relates to peptides forstimulating subcutaneous adipogenesis, wherein the peptide has a lengthof 6 to 13 amino acids and comprises a motif having the sequence:

-   -   X¹-X²-X³-X⁴-X⁵-X⁶        wherein X¹ is serine (S) or a conservative substitution        therefor; X² is a hydrophobic amino acid; X³ is any amino acid;        X⁴ is a hydrophobic amino acid; X⁵ is serine (S), arginine (R),        or a conservative substitution therefor; and X⁶ is a charged        amino acid; provided that X¹, X² and X⁴ are not all glycine (G)        and provided that X¹ is not glutamine (Q).

The length of the peptide can be, for example, 13 amino acids, 12 aminoacids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7amino acids, or 6 amino acids.

In certain aspects of the invention, X¹ is serine (S), cysteine (C),alanine (A), glycine (G), threonine (T), tyrosine (Y), or methionine(M). X¹ is preferably serine (S).

X² can be a hydrophobic nonaromatic amino acid. For example, X² can bethreonine (T), isoleucine (I), valine (V), leucine (L), alanine (A),glycine (G), cysteine (C), or methionine (M). X² is preferably threonine(T) or isoleucine (I), and is more preferably threonine (T).

X³ can be a hydrophobic nonaromatic amino acid, a hydrophobic polaramino acid, a positively charged amino acid, or proline (P). Forexample, X³ can be methionine (M), valine (V), glycine (G), threonine(T), arginine (R), leucine (L), proline (P), or a conservativesubstitution therefor. X³ is preferably methionine (M), valine (V),glycine (G), threonine (T), arginine (R), leucine (L), or proline (P).X³ is more preferably methionine (M), leucine (L), or a conservativesubstitution therefor, and is even more preferably methionine (M).

X⁴ can be a hydrophobic nonaromatic amino acid or a hydrophobic nonpolaramino acid. In some aspects of the invention, X⁴ is methionine (M),leucine (L), valine (V), or a conservative substitution therefor. X⁴ ispreferably methionine (M), leucine (L), or valine (V), and is morepreferably methionine (M).

X⁵ can be arginine (R) or a conservative substitution therefor.Alternatively, X⁵ can be serine (S) or a conservative substitutiontherefor. X⁵ is preferably serine (S).

X⁶ can be arginine (R) or a conservative substitution therefor. X⁶ ispreferably arginine (R).

In certain of the peptides for stimulating subcutaneous adipogenesis,the motif is STMMSR (SEQ ID NO: 51), SIMMSR (SEQ ID NO: 52), STLMSR (SEQID NO: 53), STVMSR (SEQ ID NO: 54), STGLSR (SEQ ID NO: 55), STTMSR (SEQID NO: 56), STRMSR (SEQ ID NO: 57), STLMRR (SEQ ID NO: 58), or STPVSR(SEQ ID NO: 59). For example, the motif can be STMMSR (SEQ ID NO: 51).

For example, the peptide is STMMSR (SEQ ID NO: 51), SIMMSR (SEQ ID NO:52), STLMSR (SEQ ID NO: 53), STVMSR (SEQ ID NO: 54), STGLSR (SEQ ID NO:55), STTMSR (SEQ ID NO: 56), STRMSR (SEQ ID NO: 57), STLMRR (SEQ ID NO:58), STPVSR (SEQ ID NO: 59), STMMSRS (SEQ ID NO: 60), STMMSRSH (SEQ IDNO: 61), STMMSRSHK (SEQ ID NO: 62), STMMSRSHKT (SEQ ID NO: 63),STMMSRSHKTR (SEQ ID NO: 64), STMMSRSHKTRS (SEQ ID NO: 65), orSTMMSRSHKTRSH (SEQ ID NO: 66). For example, the peptide can be STMMSR(SEQ ID NO: 51) or STMMSRSHK (SEQ ID NO: 62).

In certain aspects of the invention, the motif is not QLVKRK (SEQ ID NO:86), QKVLKR (SEQ ID NO: 87), GGRGRR (SEQ ID NO: 88), GGRGGR (SEQ ID NO:89), or GGGGGR (SEQ ID NO: 90).

Still another aspect of the present invention relates to peptides forstimulating subcutaneous adipogenesis, wherein the peptide has a lengthof 6 to 13 amino acids and comprises a motif having the sequence

-   -   X¹-X²-X³-X⁴-X⁵-X⁶        wherein X¹ is serine (S) or a conservative substitution therefor        and X⁶ is a charged amino acid. X²-X³-X⁴-X⁵ is an amino acid        sequence having at least 50% sequence identity with the amino        acid sequence TMMS (SEQ ID NO: 67) and having a length of 2 to 8        amino acids.

The length of the peptide can be, for example, 12 amino acids, 11 aminoacids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, or 6amino acids.

In some aspects, the length of X²-X³-X⁴-X⁵ is 3 to 7 amino acids, 3 to 6amino acids, or 4 amino acids.

X¹ is preferably serine (S).

X⁶ is preferably a positively charged amino acid, for example arginine(R), lysine (K), histidine (H), or a conservative substitution therefor.X⁶ is more preferably arginine (R) or a conservative substitutiontherefor, and is even more preferably arginine (R).

X²-X³-X⁴-X⁵ can have at least about 75% sequence identity with the aminoacid sequence SEVS (SEQ ID NO: 50).

In any of the above-described peptides, the peptide can have asubstantially alpha-helical conformation.

Any of the above-described peptides can be a beta-peptide.

Any of the above-described peptides can be in the retro form, theinverso form, or the retro-inverso form.

In any of the above-described peptides, all of the amino acids in thepeptide can be L amino acids. Alternatively, the peptide can compriseone or more D amino acids, or all of the amino acids in the peptide canbe D amino acids. Thus, the peptide can comprise a mixture of L aminoacids and D amino acids.

Peptide Variants

The peptides described herein can be composed of amino acids joined toeach other by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedstandard amino acids. The peptides may be modified by either naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques that are well known in the art. Modificationscan occur anywhere in a peptide, including the peptide backbone, theamino acid side-chains and the amino or carboxyl termini. The same typeof modification may be present in the same or varying degrees at severalsites in a peptide. Also, a peptide may contain many types ofmodifications.

In certain aspects of the invention, peptides may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclic peptides mayresult from posttranslation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine-cysteine disulfide bonds,formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, PEGylation, proteolyticprocessing, phosphorylation, prenylation, palmitoylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (see, e.g.,Creighton et al. (1993) Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York; Johnson, ed.(1983) Posttranslational Covalent Modification Of Proteins, AcademicPress, New York; Seifter et al. (1990) Meth. Enzymol., 182: 626-646;Rattan et al. (1992) Ann. N.Y. Acad. Sci., 663: 48-62; and the like).

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the peptides described herein. Such variants includedeletions, insertions, inversions, repeats, and substitutions (e.g.,conservative substitutions) selected according to general rules wellknown in the art so as have little effect on activity.

Peptoids are also contemplated wherein the peptoid has an essentiallycorresponding monomer sequence, where a natural amino acid is replacedby an N-substituted glycine derivative, if the N-substituted glycinederivative resembles the original amino acid in hydrophilicity,hydrophobicity, polarity, etc. The following are illustrative, butnon-limiting N-substituted glycine replacements:N-(1-methylprop-1-yl)glycine→isoleucine (I), N-(prop-2-yl)glycine→valine(V), N-benzylglycine→phenylalanine (F), N-(2-hydroxyethyl)glycine→serine(S), and the like. In certain aspects of the invention, substitutionsneed not be “exact”. Thus for example, in certain aspects of theinvention, N-(2-hydroxyethyl)glycine may substitute for S, T, C, and/orM; N-(2-methylprop-1-yl)glycine may substitute for V, L, and/or I;N-(2-hydroxyethyl)glycine can be used to substitute for T or S. Ingeneral, one may use an N-hydroxyalkyl-substituted glycine to substitutefor any polar amino acid, an N-benzyl- or N-aralkyl-substituted glycineto replace any aromatic amino acid, an N-alkyl-substituted glycine suchas N-butylglycine to replace any nonpolar amino acid (e.g., L, V, I,etc.), and an N-(aminoalkyl)glycine to replace any basic polar aminoacid (e.g., L and R).

Functionalization and Protecting Groups.

While the various peptides described herein are shown without protectinggroups, they can bear one or more protecting groups. The protectinggroups can be coupled to the C- and/or N-terminus of the peptide and/orto one or more internal residues of the peptide. Thus, for example, anyof the peptides described herein can bear, e.g., an acyl groupprotecting the amino terminus and/or an amide group protecting thecarboxyl terminus. These blocking groups enhance the helix-formingtendencies of the peptides.

Without being bound by a particular theory, it is believed thatblockage, particularly of the amino and/or carboxyl termini of thepeptide can improve stability and/or skin penetration when administeredtopically.

Suitable protecting groups include, but are not limited to, acetyl,amide, and alkyl groups with acetyl and alkyl groups being particularlypreferred for N-terminal protection and amide groups being preferred forcarboxyl terminal protection. Alkyl protecting groups include, but arenot limited to, alkyl chains as in fatty acids such as propionyl,formyl, and others. For example, the alkyl can be 3- to 20-carbon alkyl.Carboxyl protecting groups include amides, esters, and ether-formingprotecting groups. Such blocking groups include alkyl groups of variouslengths, e.g., groups having the formula: CH₃—(CH₂)_(n)—CO— where nranges from about 1 to about 20, preferably from about 1 to about 16 or18, more preferably from about 3 to about 13, and most preferably fromabout 3 to about 10.

Other suitable protecting groups include, but are not limited to,fluorenylmethyloxycarbonyl (FMOC), t-butoxycarbonyl (t-BOC),9-fluoreneacetyl, 1-fluorenecarboxylic, 9-florenecarboxylic,9-fluorenone-1-carboxylic, benzyloxycarbonyl, xanthyl (Xan), trityl(Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt),4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), mesitylene-2-sulphonyl(Mts), 4,4-dimethoxybenzhydryl (Mbh), tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBz1), 4-methoxybenzyl(MeOBz1), benzyloxy (BzlO), benzyl (Bzl), benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiC1-Bz1), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom), cyclohexyloxy(cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), acetyl(Ac), and trifluoroacetyl (TFA).

Protecting/blocking groups are well known to those of skill as aremethods of coupling such groups to the appropriate residue of thepeptide (see, e.g., Greene et al., (1991) Protective Groups in OrganicSynthesis, 2nd ed., John Wiley & Sons, Inc. Somerset, N.J.). Forexample, acetylation can be accomplished during synthesis when thepeptide is on the resin using acetic anhydride. Amide protection can beachieved by the selection of a proper resin for the synthesis. Duringthe synthesis of the peptide, rink amide resin can used. After thecompletion of the synthesis, the semipermanent protecting groups onacidic bifunctional amino acids such as Asp and Glu, the basic aminoacid Lys, and the hydroxyl of Tyr are all simultaneously removed. Thepeptides released from such a resin using acidic treatment have theN-terminus protected with acetyl and the carboxy-terminus protected withNH₂ with the simultaneous removal of all of the other protecting groups.

The peptides may comprise one or more D-amino acids (dextro rather thanlevo). Every other, or even every amino acid (e.g., every enantiomericamino acid) of the peptide can be a D-amino acid. For example, at least50%, at least 80%, at least 90% or even all of the enantiomeric aminoacids can be D-amino acids.

The peptides can also be functionalized with a polymer (e.g.,polyethylene glycol and/or a cellulose or modified cellulose) toincrease bioavailability.

Peptide Mimetics.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compounds are termed “peptidemimetics” or “peptidomimetics” (Fauchere (1986) Adv. Drug Res. 15: 29;Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med.Chem. 30: 1229) and are usually developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar topeptides of the invention may be used to produce an equivalenttherapeutic or prophylactic effect.

Peptidomimetics are structurally similar to a peptide (e.g., KSEVSK (SEQID NO: 3)), but have one or more peptide linkages optionally replaced bya linkage such as —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans),—COCH₂—, —CH(OH)CH₂—, or —CH₂SO— by methods known in the art and furtherdescribed in the following references: Spatola (1983) p. 267 inChemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.Weinstein, eds., Marcel Dekker, New York; Spatola (1983) Vega Data 1(3)Peptide Backbone Modifications. (general review); Morley (1980) TrendsPharm Sci pp. 463-468 (general review); Hudson et al. (1979) Int J PeptProt Res 14:177-185 (—CH₂NH—, —CH₂—CH₂—); Spatola et al. (1986) Life Sci38:1243-1249 (—CH₂—S—); Hann, (1982) J Chem Soc Perkin Trans I 307-314(—CH═CH—, cis and trans); Almquist et al. (1980) J Med Chem. 23:1392-1398 (—COCH₂—); Jennings-White et al. (1982) Tetrahedron Lett. 23:2533 (—COCH₂—); Szelke et al. (1982) European Appin. EP 45665(—CH(OH)CH₂—); Holladay et al. (1983) Tetrahedron Lett 24:4401-4404(—C(OH)CH₂—); and Hruby (1982) Life Sci., 31:189-199 (—CH₂—S—)).

A particularly useful non-peptide linkage is —CH₂NH—. Such peptidemimetics may have significant advantages over peptides, such as moreeconomical production, greater chemical stability, enhancedpharmacological properties (half-life, absorption, potency, efficacy,etc.), and/or reduced antigenicity.

Circular permutations of the peptides described herein or constrainedpeptides (including cyclized peptides) comprising the motifs describedherein or a substantially identical motif can be generated by methodsknown in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387),for example, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

Peptide Preparation.

In various aspects of the invention, the peptides described herein canbe chemically synthesized using standard chemical peptide synthesistechniques or, particularly where the peptide does not comprise D-aminoacid residues, the peptide can be recombinantly expressed. Wherepolypeptides containing one or more D-amino acids are to berecombinantly expressed, a host organism (e.g. bacteria, plant, fungalcells, etc.) can be cultured in an environment where one or more of theamino acids is provided to the organism exclusively in a D-form.Recombinantly expressed peptides in such a system then incorporate thoseD-amino acids. Also, D-amino acids can be incorporated in recombinantlyexpressed peptides using modified amino acyl-tRNA synthetases thatrecognize D-amino acids.

The peptides (containing D- and/or L-amino acids) can be chemicallysynthesized by any of a number of fluid or solid phase peptide synthesistechniques known to those of skill in the art. Solid phase synthesis inwhich the C-terminal amino acid of the sequence is attached to aninsoluble support followed by sequential addition of the remaining aminoacids in the sequence is a preferred method for the chemical synthesisof the polypeptides of the invention. Techniques for solid phasesynthesis are well known to those of skill in the art and are described,for example, by Barany and Merrifield (1963) Solid-Phase PeptideSynthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al.(1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewart et al. (1984) SolidPhase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.

The peptides can be synthesized by the solid phase peptide synthesisprocedure using a benzhydrylamine resin (Beckman Bioproducts, 0.59 mmolof NH₂/g of resin) as the solid support. The carboxy terminal amino acid(e.g., t-butylcarbonyl-Phe) is attached to the solid support through a4-(oxymethyl)phenacetyl group. This is a more stable linkage than theconventional benzyl ester linkage, yet the finished peptide can still becleaved by hydrogenation. Transfer hydrogenation using formic acid asthe hydrogen donor can be used for this purpose.

It is noted that in the chemical synthesis of peptides, particularlypeptides comprising D-amino acids, the synthesis usually produces anumber of truncated peptides in addition to the desired full-lengthproduct. Thus, the peptides are typically purified using, e.g., HPLC.

D-amino acids, beta amino acids, non-natural amino acids, non-standardamino acids, and the like can be incorporated at one or more positionsin the peptide simply by using the appropriately derivatized amino acidresidue in the chemical synthesis. Modified residues for solid phasepeptide synthesis are commercially available from a number of suppliers(see, e.g., Advanced Chem Tech, Louisville; Nova Biochem, San Diego;Sigma, St Louis; Bachem California Inc., Torrance, etc.). The D-formand/or otherwise modified amino acids can be completely omitted orincorporated at any position in the peptide as desired.

When the peptides are synthesized using recombinant expression systems,a DNA sequence that encodes the desired peptide is typically created,and placed in an expression cassette under the control of a promoter toexpress the peptide in a host cell. The expressed peptide is isolatedand, if required, renatured. DNA encoding a peptide of the invention canbe prepared by any suitable method, including, for example, cloning andrestriction of appropriate sequences or direct chemical synthesis. Thisnucleic acid can be easily ligated into an appropriate vector containingappropriate expression control sequences (e.g. promoter, enhancer,etc.), and, optionally, containing one or more selectable markers (e.g.antibiotic resistance genes).

A nucleic acid sequence encoding a peptide described herein can beexpressed in a variety of host cells, including, but not limited to, E.coli, other bacterial hosts, yeast, fungus, and various highereukaryotic cells such as insect cells (e.g. SF3), the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene willtypically be operably linked to appropriate expression control sequencesfor each host. For E. coli this can include a promoter such as the T7,trp, or lambda promoters, a ribosome binding site and preferably atranscription termination signal. For eukaryotic cells, illustrativecontrol sequences can include a promoter and often an enhancer (e.g., anenhancer derived from immunoglobulin genes, SV40, cytomegalovirus,etc.), and a polyadenylation sequence, and may include splice donor andacceptor sequences.

The plasmids can be transferred into the chosen host cell by well-knownmethods such as calcium chloride transformation for E. coli and calciumphosphate treatment or electroporation for mammalian cells. Cellstransformed by the plasmids can be selected by resistance to antibioticsconferred by genes contained on the plasmids, such as the amp, gpt, neoand hyg genes.

Once expressed, the recombinant peptide(s) can be purified according tostandard procedures of the art, including, but not limited to, ammoniumsulfate precipitation, affinity columns, column chromatography, and gelelectrophoresis (see, generally, R. Scopes, (1982) Protein Purification,Springer-Verlag, N.Y.; Deutscher (1990) Methods in Enzymology Vol. 182:Guide to Protein Purification., Academic Press, Inc. N.Y.).Substantially pure compositions of at least about 90 to 95% homogeneityare preferred, and 98 to 99% or more homogeneity are most preferred.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the peptide may possess aconformation substantially different than desired native conformation.In this case, it may be necessary to denature and reduce the peptide andthen to cause the molecule to re-fold into the preferred conformation.Methods of reducing and denaturing proteins and inducing re-folding arewell known to those of skill in the art (see, e.g., Debinski et al.(1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993)Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski et al., for example, describes thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The protein is then refolded in a redox buffer containing oxidizedglutathione and L-arginine.

One of skill would recognize that modifications can be made to thepeptides of the invention without diminishing their biological activity.Some modifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

Pharmaceutical and Cosmetic Formulations

The peptide(s) described herein are administered to a subject (e.g., amammal) in need thereof, e.g., to induce or potentiate subcutaneousadipocyte recruitment or formation (e.g., to induce subcutaneousadipogenesis).

These peptide(s) can be administered in the “native” form or, ifdesired, in the form of salts, esters, amides, prodrugs, derivatives,and the like, provided the salt, ester, amide, prodrug or derivative issuitable pharmacologically, i.e., effective in the present method(s).Salts, esters, amides, prodrugs and other derivatives of the peptide(s)can be prepared using standard procedures known to those skilled in theart of synthetic organic chemistry and described, for example, by March(1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure,4th Ed. N.Y. Wiley-Interscience.

Methods of formulating such derivatives are known to those of skill inthe art. For example, the disulfide salts of a number of delivery agentsare described in PCT Publication WO 2000/059863. Similarly, acid saltsof therapeutic peptides, peptoids, or other mimetics, and can beprepared from the free base using conventional methodology thattypically involves reaction with a suitable acid. Generally, the baseform of the peptide is dissolved in a polar organic solvent such asmethanol or ethanol and the acid is added thereto. The resulting salteither precipitates or can be brought out of solution by addition of aless polar solvent. Suitable acids for preparing acid addition saltsinclude, but are not limited to, both organic acids, e.g., acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid,malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, or salicylic acid, aswell as inorganic acids, e.g., hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, or phosphoric acid. An acid addition saltcan be reconverted to the free base by treatment with a suitable base.Certain particularly preferred acid addition salts of the peptidesdescribed herein include halide salts, such as may be prepared usinghydrochloric or hydrobromic acids. Conversely, preparation of basicsalts of the peptides s described herein are prepared in a similarmanner using a pharmaceutically acceptable base such as sodiumhydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide,or trimethylamine. Basic salts include alkali metal salts, e.g., thesodium salt, and copper salts.

For the preparation of salt forms of a peptide, the pKa of thecounterion is preferably at least about 2 pH lower than the pKa of theagent. Similarly, for the preparation of salt forms of acidic agent, thepKa of the counterion is preferably at least about 2 pH greater than thepKa of the agent. This permits the counterion to bring the solution's pHto a level lower than the pH_(max) to reach the salt plateau, at whichthe solubility of salt prevails over the solubility of free acid orbase. The generalized rule of difference in pKa units of the ionizablegroup in the active pharmaceutical ingredient (API) and in the acid orbase is meant to make the proton transfer energetically favorable. Whenthe pKa of the API and counterion are not significantly different, asolid complex may form but may rapidly disproportionate (i.e., breakdown into the individual entities of peptide and counterion) in anaqueous environment.

Typically, the counterion is a pharmaceutically acceptable counterion.Suitable anionic salt forms include, but are not limited to, acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

Preparation of peptide esters typically involves functionalization ofhydroxyl and/or carboxyl groups that are present within the molecularstructure of the peptide. For example, the esters are typicallyacyl-substituted derivatives of free alcohol groups, i.e., moieties thatare derived from carboxylic acids of the formula RCOOH where R is alkyl,and preferably is lower alkyl. Esters can be reconverted to the freeacids, if desired, by using conventional hydrogenolysis or hydrolysisprocedures.

Amides of the peptides can also be prepared using techniques known tothose skilled in the art. For example, amides can be prepared fromesters, using suitable amine reactants, or they can be prepared from ananhydride or an acid chloride by reaction with ammonia or a lower alkylamine.

The peptides can be formulated for subcutaneous, parenteral, topical,oral, nasal (or otherwise inhaled), rectal, or local administration,such as by aerosol or transdermally. The compositions can beadministered in a variety of unit dosage forms depending upon the methodof administration. Suitable unit dosage forms, include, but are notlimited to powders, tablets, pills, capsules, lozenges, suppositories,patches, nasal sprays, injectables, implantable sustained-releaseformulations, lipid complexes, etc.

The peptides described herein can also be combined with apharmaceutically or cosmetically acceptable carrier (excipient) to forma pharmacological or cosmetic composition. Pharmaceutically acceptablecarriers include those approved by a regulatory agency of the Federal ora state government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in or on animals, and more particularlyhumans. A “carrier” refers to, for example, a diluent, adjuvant,excipient, auxiliary agent or vehicle with which one or more of thepeptides described herein is administered.

Pharmaceutically acceptable carriers can contain one or morephysiologically acceptable compounds that act, for example, to stabilizethe composition or to increase or decrease the absorption of thepeptide. Physiologically acceptable compounds can include, for example,carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins, protection and uptake enhancers such as lipids, compounds thatreduce the clearance or hydrolysis of the peptides, or other excipients,stabilizers and/or buffers.

Other physiologically acceptable compounds, particularly of use in thepreparation of tablets, capsules, gel caps, and the like include, butare not limited to binders, diluent/fillers, disentegrants, lubricants,and suspending agents.

To manufacture an oral dosage form (e.g., a tablet), an excipient (e.g.,lactose, sucrose, starch, mannitol, etc.), an optional disintegrator(e.g. calcium carbonate, carboxymethylcellulose calcium, sodium starchglycollate, crospovidone etc.), a binder (e.g. alpha-starch, gum arabic,microcrystalline cellulose, carboxymethylcellulose,polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.), andan optional lubricant (e.g., talc, magnesium stearate, polyethyleneglycol 6000, etc.), for instance, are added to the peptide and theresulting composition is compressed. Where necessary, the compressedproduct is coated using known methods for masking taste or for entericdissolution or sustained release. Suitable coating materials include,but are not limited to ethyl-cellulose, hydroxymethylcellulose,polyoxyethylene glycol, cellulose acetate phthalate,hydroxypropylmethylcellulose phthalate, and EUDRAGIT (Rohm & Haas,Germany; methacrylic-acrylic copolymer).

Other physiologically acceptable compounds that can be formulated withthe peptides include wetting agents, emulsifying agents, dispersingagents or preservatives that are particularly useful for preventing thegrowth or action of microorganisms. Various preservatives are well knownand include, for example, phenol and ascorbic acid. One skilled in theart would appreciate that the choice of pharmaceutically acceptablecarrier depends, for example, on the route of administration of thepeptide and on the particular physio-chemical characteristics of thepeptide.

Preferably, the excipients are sterile and generally free ofcontaminants. These compositions can be sterilized by conventional,well-known sterilization techniques. For various oral dosage formexcipients, such as tablets and capsules, sterility is not required; theUSP/NF standard is usually sufficient.

Nanoemulsion Formulations.

The peptides described herein can be formulated in a nanoemulsion.Nanoemulsions include, but are not limited to, oil-in-water (O/W)nanoemulsions, and water-in-oil (W/O) nanoemulsions. Nanoemulsions canbe defined as emulsions with mean droplet diameters ranging from about20 to about 1000 nm. Usually, the average droplet size is between about20 nm or 50 nm and about 500 nm. The terms sub-micron emulsion (SME) andmini-emulsion are used as synonyms.

Illustrative oil-in-water (O/W) nanoemulsions include, but are notlimited to: (1) surfactant micelles, which are micelles composed ofsmall molecules, surfactants or detergents (e.g., SDS/PBS/2-propanol)which are suitable for predominantly hydrophobic peptides; (2) polymermicelles, which are micelles composed of polymer, copolymer, or blockcopolymer surfactants (e.g., Pluronic L64/PBS/2-propanol) which aresuitable for predominantly hydrophobic peptides; (3) blended micelles,which are micelles in which there is more than one surfactant componentor in which one of the liquid phases (generally an alcohol (e.g.,ethanol) or fatty acid compound) participates in the formation of themicelle (e.g., octanoic acid/PBS/EtOH) which are suitable forpredominantly hydrophobic peptides; (4) integral peptide micelles, whichare blended micelles in which the peptide serves as an auxiliarysurfactant, forming an integral part of the micelle (e.g., amphipathicpeptide/PBS/mineral oil) which are suitable for amphipathic peptides;and (5) pickering (solid phase) emulsions, which are emulsions in whichthe peptides are associated with the exterior of a solid nanoparticle(e.g., polystyrene nanoparticles/PBS/no oil phase) which are suitablefor amphipathic peptides.

Illustrative water-in-oil (W/O) nanoemulsions include, but are notlimited to: (1) surfactant micelles, which are micelles composed ofsmall molecules surfactants or detergents (e.g., dioctylsulfosuccinate/PBS/2-propanol, isopropylmyristate/PBS/2-propanol, etc.)which are suitable for predominantly hydrophilic peptides; (2) polymermicelles, which are micelles composed of polymer, copolymer, or blockcopolymer surfactants (e.g., PLURONIC® L121/PBS/2-propanol), which aresuitable for predominantly hydrophilic peptides; (3) blended micelles,which are micelles in which there is more than one surfactant componentor in which one of the liquid phases (generally an alcohol (e.g.,ethanol) or fatty acid compound) participates in the formation of themicelle (e.g., capric/caprylic diglyceride/PBS/EtOH) which are suitablefor predominantly hydrophilic peptides; (4) integral peptide micelles,which are blended micelles in which the peptide serves as an auxiliarysurfactant, forming an integral part of the micelle (e.g., amphipathicpeptide/PBS/polypropylene glycol) which are suitable for amphipathicpeptides; and (5) pickering (solid phase) emulsions, which are emulsionsin which the peptides are associated with the exterior of a solidnanoparticle (e.g., chitosan nanoparticles/no aqueous phase/mineral oil)which are suitable for amphipathic peptides.

As indicated above, the nanoemulsions can comprise one or moresurfactants or detergents. For example, the surfactant can be anon-anionic detergent (e.g., a polysorbate surfactant or apolyoxyethylene ether). Surfactants that find use in the presentinvention include, but are not limited to, surfactants such as theTWEEN®, TRITON®, and TYLOXAPOL® families of compounds. Preferably, thesurfactant is a polysorbate surfactant (e.g., TWEEN 20®, TWEEN 40®,TWEEN 60®, and TWEEN 80®, a phenoxypolyethoxyethanol (e.g., TRITON®X-100, X-301, X-165, X-102, and X-200, and TYLOXAPOL®), or sodiumdodecyl sulfate.

The nanoemulsion can further comprise an emulsifying agent to aid in theformation of the emulsion. Emulsifying agents include compounds thataggregate at the oil/water interface to form a kind of continuousmembrane that prevents direct contact between two adjacent droplets.Some oil-in-water emulsion compositions can readily be diluted withwater to a desired concentration without impairing their anti-pathogenicproperties.

In addition to discrete oil droplets dispersed in an aqueous phase,oil-in-water emulsions can also contain other lipid structures, such assmall lipid vesicles (e.g., lipid spheres that often consist of severalsubstantially concentric lipid bilayers separated from each other bylayers of aqueous phase), micelles (e.g., amphiphilic molecules in smallclusters of 50-200 molecules arranged so that the polar head groups faceoutward toward the aqueous phase and the apolar tails are sequesteredinward away from the aqueous phase), or lamellar phases (lipiddispersions in which each particle consists of parallel amphiphilicbilayers separated by thin films of water). These lipid structures areformed as a result of hydrophobic forces that drive apolar residues(e.g., long hydrocarbon chains) away from water. The lipid preparationscan generally be described as surfactant lipid preparations (SLPs). SLPsare minimally toxic to mucous membranes and are believed to bemetabolized within the small intestine (see e.g., Hamouda et al., (1998)J. Infect. Disease 180: 1939).

The emulsion can comprise a discontinuous oil phase distributed in anaqueous phase, a first component comprising an alcohol (e.g., ethanol)and/or glycerol, and a second component comprising a surfactant or ahalogen-containing compound. The aqueous phase can comprise any type ofaqueous phase including, but not limited to, water (e.g., deionizedwater, distilled water, water-for-injection, tap water) and solutions(e.g., phosphate buffered saline solution, or other buffer systems). Theoil phase can comprise any type of oil including, but not limited to,plant oils (e.g., soybean oil, avocado oil, flaxseed oil, coconut oil,cottonseed oil, squalene oil, olive oil, canola oil, corn oil, rapeseedoil, safflower oil, and sunflower oil), animal oils (e.g., fish oil),flavor oil, water insoluble vitamins, or mineral oil. Generally, the oilphase comprises about 30 to about 90 volume % of the oil-in-wateremulsion (i.e., constitutes 30-90% of the total volume of the finalemulsion), more preferably from about 50 to about 80 volume % of theemulsion.

The emulsion can also include a halogen-containing compound such as achloride salt (e.g., NaCl, KCl, etc.), a cetylpyridinium halide, acetyltrimethylammonium halide, a cetyldimethylethylammonium halide, acetyldimethylbenzylammonium halide, a cetyltributylphosphonium halide, adodecyltrimethylammonium halide, a tetradecyltrimethylammonium halide,cetylpyridinium chloride, cetyltrimethylammonium chloride,cetylbenzyldimethylammonium chloride, cetylpyridinium bromide,cetyltrimethylammonium bromide, cetyldimethylethylammonium bromide,cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide, ortetradecyltrimethylammonium bromide.

The emulsion can also include a quaternary ammonium compound such asN-alkyldimethyl benzyl ammonium saccharinate;1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol; 1-Decanaminium,N-decyl-N,N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride;2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride; alkyl-1- or3-benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride; alkylbis(2-hydroxyethyl)benzyl ammonium chloride; alkyl demethyl benzylammonium chloride; alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride(100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (50%C14, 40% C12, 10% C16); alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (55% C14, 23% C12, 20% C16); alkyl dimethyl benzyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (100% C14); alkyldimethyl benzyl ammonium chloride (100% C16); alkyl dimethyl benzylammonium chloride (41% C14, 28% C12); alkyl dimethyl benzyl ammoniumchloride (47% C12, 18% C14); alkyl dimethyl benzyl ammonium chloride(55% C16, 20% C14); alkyl dimethyl benzyl ammonium chloride (58% C14,28% C16); alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12);alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14); alkyldimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyl dimethylbenzyl ammonium chloride (65% C12, 25% C14); alkyl dimethyl benzylammonium chloride (67% C12, 24% C14); alkyl dimethyl benzyl ammoniumchloride (67% C12, 25% C14); alkyl dimethyl benzyl ammonium chloride(90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride (93% C14, 4%C12); alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18); alkyldimethyl benzyl ammonium chloride (and) didecyl dimethyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids);alkyl dimethyl benzyl ammonium chloride (C12-C16); alkyl dimethyl benzylammonium chloride (C12-C18); alkyl dimethyl benzyl and dialkyl dimethylammonium chloride; alkyl dimethyl dimethybenzyl ammonium chloride; alkyldimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyldimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as inthe fatty acids of soybean oil); alkyl dimethyl ethylbenzyl ammoniumchloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14); alkyldimethyl isoproylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3%C18); alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1%C12); alkyl trimethyl ammonium chloride (90% C18, 10% C16);alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18); Di-(C8-10)-alkyldimethyl ammonium chlorides; dialkyl dimethyl ammonium chloride; dialkyldimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkylmethyl benzyl ammonium chloride; didecyl dimethyl ammonium chloride;diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammoniumchloride; dodecyl bis(2-hydroxyethyl) octyl hydrogen ammonium chloride;dodecyl dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyldimethyl benzyl ammonium chloride; heptadecyl hydroxyethylimidazoliniumchloride; hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine;myristalkonium chloride and/or quaternium-14;N,N-dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethylbenzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl ammoniumchloride; n-tetradecyl dimethyl benzyl ammonium chloride monohydrate;octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammoniumchloride; octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride;oxydiethylenebis (alkyl dimethyl ammonium chloride); dicocoalkyldimethyl, chloride; trimethoxysily propyl dimethyl octadecylammonium chloride; trimethoxysilyl quats, trimethyl dodecylbenzylammonium chloride; n-dodecyl dimethyl ethylbenzyl ammonium chloride;n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyl dimethylbenzyl ammonium chloride; n-tetradecyl dimethyl ethylbenzyl ammoniumchloride; or n-octadecyl dimethyl benzyl ammonium chloride.

Nanoemulsion formulations and methods of making such are well known tothose of skill in the art and described for example in U.S. Pat. Nos.7,476,393, 7,468,402, 7,314,624, 6,998,426, 6,902,737, 6,689,371,6,541,018, 6,464,990, 6,461,625, 6,419,946, 6,413,527, 6,375,960,6,335,022, 6,274,150, 6,120,778, 6,039,936, 5,925,341, 5,753,241,5,698,219, an d5,152,923 and in Fanun et al. (2009) Microemulsions:Properties and Applications (Surfactant Science), CRC Press, Boca RatonFl.

Cosmetic Formulations.

One or more of the peptides described herein can be incorporated intoformulations for cosmetic use. Such cosmetic formulations can be fortopical application and can be formulated as skin creams (e.g., facecreams) or body lotions, wrinkle-removing creams, or incorporated intocosmetics, sunscreens, or moisturizers.

The peptides can be incorporated into formulations that optionallyfurther include fillers, moisturizers, vitamins (e.g., vitamin E),and/or colorants/tints.

Suitable injectable cosmetic formulations include, but are not limitedto, formulations incorporating one or more of the peptides incombination with one or more filler materials. Illustrative materialsusable as injectable cosmetic wrinkle fillers include, but are notlimited to, temporary (absorbable) fillers such as collagen (e.g.,synthetic collagen, bovine collagen, porcine collagen, human collagen,etc.), hyaluronic acid gel, calcium hydroxylapatite (typically implantedin the form of a gel), or poly-L-lactic acid (PLLA). The peptides canalso be incorporated into injectable cosmetic formulations containingpermanent (non-absorbable) fillers. Illustrative “permanent” fillersinclude, but are not limited to, polymethylmethacrylate beads (PMMAmicrospheres).

The peptides described herein can be incorporated into or administeredin conjunction with commercial dermal fillers (e.g., RADIESSE®volumizing filler (about 30% by volume calcium hydroxylapatite (CaHA)microspheres (diameter of 25 μm to 45 μm) suspended in a sodiumcarboxymethylcellulose gel carrier)), JUVEDERM® injectable gel (acrosslinked hyaluronic acid produced by Streptococcus equi bacteria,formulated to a concentration of 24 mg/mL, optionally with 0.3% w/wlidocaine, in a physiologic buffer), RESTYLANE® dermal filler (a gel of20 mg/mL hyaluronic acid generated by Streptococcus species of bacteria,chemically crosslinked with BDDE, and suspended in phosphate bufferedsaline), SCULPTRA® Aesthetic injectable implant (suspension containingmicroparticles of poly-L-lactic acid (PLLA), carboxymethylcellulose,non-pyrogenic mannitol and sterile water for injection).

Such injectable formulations can additionally include an anesthetic(e.g., lidocaine or an analog thereof).

The injectable formulations are substantially sterile or sterile and/ormeet regulatory agency guidelines for subcutaneous injectable fillers

Dose/Administration

The peptides described herein can be administered to a subject using anyroute known in the art, including e.g., by injection (e.g., intravenous,intraperitoneal, subcutaneous, intramuscular, or intradermal),inhalation, transdermal application, rectal administration, vaginaladministration, or oral administration. Preferred routes ofadministration include subcutaneous, transdermal, or topicalapplication.

An effective amount of the peptides can be administered via local (i.e.,non-systemic) administration, such as by peripheral administration whichincludes, but is not limited to, peripheral intramuscular,intraglandular, and subcutaneous administration.

Administration of the peptide(s) can be in any convenient manner, e.g.,by injection, intravenous and arterial stents (including elutingstents), catheter, oral administration, inhalation, transdermalapplication, rectal administration, and the like.

The peptides can be formulated with a pharmaceutically acceptablecarrier, e.g., as described above, prior to administration.Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations for the peptides described herein (see,e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

The dose administered to a subject, in the context of the methodsdescribed herein should be sufficient to effect a beneficial therapeuticresponse (e.g., increased subcutaneous adipogenesis) in the subject overtime. The dose will be determined by the efficacy of the particularvehicle/delivery method employed, the site of administration, the routeof administration, and the condition of the subject, as well as the bodyweight or surface area of the subject to be treated. The size of thedose also will be determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularpeptide in a particular subject.

The terms “effective amount” or “amount effective to” or“therapeutically effective amount” refers to an amount sufficient toinduce a detectable therapeutic (or cosmetic) response in the subject(e.g. an amount sufficient to induce or to potentiate adipogenesis). Indetermining the effective amount of peptide to be administered, theattending physician will decide the dosage with which to treat eachindividual patient, taking into consideration a variety of factors, suchas age, body weight, general health, diet, sex, peptide and formulationto be administered, route of administration, the severity of thecondition being treated and the response of the subject and any adverseeffects (e.g., irritation or allergies). The peptides described hereincan also be administered at a rate determined by the LD₅₀ of the peptideand/or the therapeutic efficacy/activity of the peptide, and theside-effects of the peptide at various concentrations, as applied to themass and overall health of the subject.

The dose of peptide can vary widely, and will be selected primarilybased on activity of the active ingredient, and body weight inaccordance with the particular mode of administration selected and thesubject's needs. Concentrations, however, will typically be selected toprovide dosages ranging from about 0.1 to about 50 mg/kg/day or more.Typical dosages range from about 1 mg/kg/day to about 50 mg/kg/day,about 2 mg/kg/day to about 30 mg/kg/day, or about 3 mg/kg/day to about20 mg/kg/day, such as about 3 mg/kg/day to about 3.5 mg/kg/day,preferably from about 3.5 mg/kg/day to about 7.2 mg/kg/day, morepreferably from about 7.2 mg/kg/day to about 11.0 mg/kg/day, or mostpreferably from about 11.0 mg/kg/day to about 15.0 mg/kg/day. Dosages ofpeptides can range from about 10 mg/kg/day to about 50 mg/kg/day.Alternatively, the dosages can range from about 20 mg to about 50 mggiven twice daily. It will be appreciated that such dosages may bevaried to optimize a therapeutic and/or cosmetic regimen in a particularsubject or group of subjects.

Administration can be accomplished via single or divided doses, e.g.,doses administered on a regular basis (e.g., once daily or twice daily)for a period of time (e.g., 2, 3, 4, 5, or 6 days or 1-3 weeks or more).

The peptides described herein can be administered systemically (e.g.,orally, or as an injectable) in accordance with standard methods wellknown to those of skill in the art. Peptides can be administered to theoral cavity in various forms such as lozenges, aerosol sprays,mouthwash, coated swabs, and the like. Various buccal, and sublingualformulations are also contemplated. The peptides can be administered ina depot formulation when formulated as an injectable to providetreatment over a period of time.

The peptides can be administered topically, e.g., to the skin surface,to a topical lesion or wound, to a surgical site, and the like.

The peptides can also be delivered through the skin using conventionaltransdermal drug delivery systems, i.e., transdermal “patches” whereinthe peptides are typically contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the drug composition is typically contained in a layer, or“reservoir,” underlying an upper backing layer. The term “reservoir” inthis context refers to a quantity of active ingredient that isultimately available for delivery to the surface of the skin. Thus, forexample, the “reservoir” may include the active ingredient peptide in anadhesive on a backing layer of the patch, or in any of a variety ofdifferent matrix formulations known to those of skill in the art. Thepatch may contain a single reservoir, or it may contain multiplereservoirs. For example, the reservoir can comprise a polymeric matrixof a pharmaceutically acceptable contact adhesive material that servesto affix the system to the skin during drug delivery. Examples ofsuitable skin contact adhesive materials include, but are not limitedto, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, for example, thedrug-containing reservoir and skin contact adhesive are present asseparate and distinct layers, with the adhesive underlying the reservoirwhich, in this case, may be either a polymeric matrix as describedabove, or it may be a liquid or hydrogel reservoir, or may take someother form. The backing layer in these laminates, which serves as theupper surface of the device, preferably functions as a primarystructural element of the “patch” and provides the device with much ofits flexibility. The material selected for the backing layer ispreferably substantially impermeable to the peptides and any othermaterials that are present.

Other formulations for topical delivery include, but are not limited to,ointments, gels, sprays, fluids, and creams. Ointments are semisolidpreparations that are typically based on petrolatum or other petroleumderivatives. As with other carriers or vehicles, an ointment base shouldbe inert, stable, nonirritating and nonsensitizing. Creams containingthe selected peptide are typically viscous liquid or semisolidemulsions, often either oil-in-water or water-in-oil. Cream bases aretypically water-washable, and contain an oil phase, an emulsifier and anaqueous phase. The oil phase, also sometimes called the “internal”phase, is generally comprised of petrolatum and a fatty alcohol such ascetyl or stearyl alcohol; the aqueous phase usually, although notnecessarily, exceeds the oil phase in volume, and generally contains ahumectant. The emulsifier in a cream formulation is generally anonionic, anionic, cationic or amphoteric surfactant. The specificointment or cream base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for optimum drug delivery.

One or more peptides described herein can be provided as a“concentrate”, e.g., in a storage container (e.g., in a premeasuredvolume) ready for dilution, or in a soluble capsule ready for additionto a volume of water, alcohol, hydrogen peroxide, or other diluent. Forexample, the peptides can be lyophilized for later reconstitution.

Illustrative Uses

The adipogenic peptides (or mimetics thereof) described herein find usein a number of applications. For example, enhancing the formation ofsubcutaneous fat has use in plastic surgery procedures sincesubcutaneous fat provides plumpness and firmness to skin. Aging skincontains less subcutaneous fat. Therefore administering one or morepeptides described herein to the desired area to promote subcutaneousfat formation results in plumper and more youthful appearing skin. Thisapproach can replace current methods for transplanting adipocytes fromother areas of the body (e.g., the thigh or buttocks), a procedure thatoften exhibits a low success rate.

The peptides described herein can be administered to selectively enhancesubcutaneous adipose tissue (e.g., to enhance subcutaneous adiposetissue without substantially increasing visceral adipose and/or otheradipose tissue). In response to administration of the peptides,adipocyte formation occurs in dermal fibroblasts and volume is added ina selected subcutaneous area in the subject.

The peptides described herein can be used to reducing scarring. This canbe accomplished by administering one or more of the peptides in anamount sufficient to reduce the area of scarring, and/or to improve theappearance of a scarred area. The scarring, for example, can be scarringproduced by a burn, scarring produced by surgery, scarring produced byacne, scarring produced by a biopsy, or scarring produced by an injury.

The peptides described herein can be used, e.g., in various cosmeticprocedures, to improve the appearance of skin. This can be accomplishedby administering one or more of the peptides in an amount sufficient toimprove the appearance of skin in an area of a subject. Suchadministering can include subcutaneously administration to a region suchas lips, eye lids, cheeks, forehead, chin, neck, and the like. Thepeptides can be used in these methods, or others to reduce wrinkles,reduce sagging skin, improve the surface texture of skin, diminish,remove or fill-in wrinkles, remove or diminish age spots, and/or removedark circles under the eyes. These cosmetic applications areillustrative and not intended to be limiting. In view of the teachingsprovided herein, other cosmetic applications will be recognized by andavailable to one of skill in the art.

The peptides described herein can be used to improve tissue volume in anarea of a subject. This can be accomplished by administering one or moreof the peptides described herein in an amount sufficient to increasetissue volume in an area of a subject. For example, the increase oftissue volume can involve firming or augmenting breast tissue and/orfirming or augmenting tissue in the buttocks or other regions of thebody or face.

The peptides can also be used to smooth skin in an area of a subject.This can be accomplished by administering one or more of the peptidesdescribed herein in an amount sufficient to smooth skin in the desiredarea. The smoothing can include smoothing skin scarred by acne,smoothing areas of cellulite, smoothing or reducing stretch marks,and/or smoothing out wrinkles.

The peptides described herein can be used to recruit stem cells to theformation of subcutaneous fat in a subject. This can be accomplished byadministering one or more of the peptides described herein in an amountsufficient to recruit stem cells to the formation of subcutaneous fat.This has utility, for example, in various reconstructive surgicalprocedures and the like.

The peptides described herein can be used to reconstructing tissue in asubject. Such reconstruction can include, for example, breastreconstruction (e.g. after surgery to remove tumors), or face or limbreconstruction (e.g. after car accident or burning). This can beaccomplished by administering one or more of the peptides describedherein in an amount to increase the volume of the tissue during or aftera tissue reconstruction procedure. The peptides are optionally used inconjunction with tissue grafting material or other procedures thatenhance youthful skin or repair of damaged tissues.

The peptides can also be used to reduce heel pain in a subject byadministering one or more of the peptides described herein in an amountsufficient to reduce heel pain experienced by the subject when walking.

The peptides described herein can be administered for augmentation ofsubcutaneous fat to improve thermo regulation and/or improve immunefunction. The subject can be treated with the peptides to preventdisease or to treat ongoing disease associated with increased organ fatincluding but not limited to cardiovascular disease, and other obesityassociated diseases.

The administration in any of these methods can be local or systemic, andcan be by any route described herein, such as topical, subcutaneous,transdermal, oral, nasal, vaginal, and/or rectal administration.Preferably, the peptides are administered by subcutaneous injection.Alternatively, the peptides are preferably administered topically in theform of a skin cream such as a face cream, or transdermally via atransdermal patch.

While the uses and methods are described with respect to use in humans,they are also suitable for animal, e.g., veterinary use. Thus certainpreferred organisms include, but are not limited to humans, non-humanprimates, canines, equines, felines, porcines, ungulates, largomorphs,and the like.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1: Assays to Measure Adipogenesis

The following assays were used to assess adipogenic activity of thepeptides in the experiments described below in Example 2.

Assay 1

Reagents: (1) 3-isobutyl-1-methylxanthine (IBMX) solution: 0.5M in DMSO;(2) Insulin solution: 10 mg/mL recombinant human insulin; (3)Dexamethasone solution in 10 mM in ethanol; (4) Oil Red 0 (0.36% in 60%isopropanol) or BODIPY dye (BODIPY 493/503(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionicacid, succinimidyl ester) from Invitrogen at 4-10 μM).

Preparation of Media:

Adipogenesis Initiation Media: mesenchymal stem cell expansion media/0.5mM IBMX/1 μM dexamethasone. Mesenchymal stem cell expansion media iscommercially available from Millipore. The IBMX Solution was diluted1:1000 and the Dexamethasone Solution was diluted 1:10,000 in expansionmedia containing antibiotics and antimycotics. The prepared media wasstored at 4° C.

Adipogenesis Progression Media for positive control: mesenchymal stemcell expansion media/10 μg/mL insulin. The Insulin Solution was diluted1:1000 in the expansion media and stored at 4° C.

Experimental Adipogenesis Progression Media: mesenchymal stem cellexpansion media/1-100 μg/mL RHAMM antagonist peptides.

Adipogenesis Maintenance Media: mesenchymal stem cell expansion mediacontaining antibiotics and antimycotics.

Negative Control Media: DMEM/10% normal calf serum.

Assay Method:

Pre-adipocyte stem cells including bone marrow mesenchymal stem cellsand fibroblasts including reticular and papillary dermal fibroblastswere propagated using standard tissue culture methods. Cells weretrypsinized to remove the cells from the substratum. The trypsin wasneutralized and cells were counted using a hemocytometer. Cells wereresuspended at 30,000 cells/mL in expansion media. 1 mL of cellsuspension was plated per well of 24-well plate, with several wells leftempty for blank well staining.

Cells were incubated for 1-2 days until they reached confluency.Approximately 1.8 mL of media was removed from each well and replacedwith 2 mL Adipogenesis Initiation Media. For negative control wells,expansion media only was used at this step and for subsequent mediachanges. For all media changes, media was replaced as gently as possibleto avoid disturbing the monolayer.

Cells were incubated for 48 hours at 37° C., 5% CO₂. 2 mL media wasremoved from each well, and replaced with 2 mL either positive controlor experimental Adipogenesis Progression Media per well. Cells wereagain incubated for 48 hours at 37° C., 5% CO₂. 2 mL of media wasremoved from each well and replaced with 2 mL Adipogenesis MaintenanceMedia per well. In addition, media was added to the empty wells reservedfor blank well staining Cells were incubated for at least 48 hours at37° C., 5% CO₂. Media was replaced every 48-72 hours. Accumulation ofintracellular lipid droplets continued for at least 5 days.

Quantification of Adipogenesis:

Oil Red O Staining

For quantification of Oil Red 0 staining, media was removed and cellswere washed twice with PBS, using care to gently add the PBS. 0.5 mL OilRed 0 solution was added per well of 24-well plate, including to thewells lacking cells, but containing media. Cells were incubated for 15minutes at room temperature. The staining solution was then removed andwells were washed three times with 1 mL of phosphate buffered saline(PBS). In some experiments, following removal of the last wash, stainedplates were scanned or photographed.

Adipogenesis was quantified using a spectrophotometer or fluorometer.0.25 mL Dye Extraction Solution (isopropyl alcohol) was added per well,and the plate was set on an orbital shaker or platform rocker for 15-30minutes. Extracted dye was transferred into a cuvette and absorbance wasread in a spectrophotometer at visible range for oil red 0 ortransferred to a 96-well plate and quantified in a plate reader.

BODIPY Staining

For quantification of BODIPY dye uptake, media was removed and cellswere washed twice with PBS, using care to gently add the PBS. Cells werethen fixed in 3% paraformaldehyde, washed in PBS then incubated for 1hour in the dark with the BODIPY solution, prepared in PBS. Monolayerswere washed and then extracted for measurement of dye in a fluorometeror mounted on slides and examined in a confocal fluorescent microscope.

Blank Well Staining

The stain extracted from wells lacking cells represents non-specificbinding of the dye to the plate. This value was subtracted from theabsorbances of experimental wells to obtain a more accurate assessmentof specific staining

Assay 2

Reagents:

The dexamethasone, insulin, and Oil Red 0 solutions were the same asdescribed above for assay 1. Indomethacin Solution contained 10 mMindomethacin in methanol. The BODIPY 493/503 dye was used at aconcentration of 4 μM.

Preparation of Media:

Adipogenesis Maintenance Media was prepared as described above.

Adipogenesis Induction Media: To prepare 501 mL of AdipogenesisInduction Media, 50 μL of the 10 mM stock solution of dexamethasone, 500μL of the 10 mg/ml stock solution of insulin, 5 ml of a 10 mM stocksolution of indomethacin, and 5 mL of a 100× stock solution ofpenicillin and streptomycin were added to 490 mL of mesenchymal stemcell expansion media.

Assay Method:

Pre-adipocyte stem cells, bone marrow mesenchymal stem cells andfibroblasts such as dermal reticular and papillary fibroblasts werepropagated using standard tissue culture methods. Cells were trypsinizedto remove cells from the substratum, the trypsin was neutralized, andcells were counted using a hemocytometer. Cells were resuspended at60,000 cells/ml in low glucose DMEM containing 10% calf serum. 1 mL ofcell suspension per well was plated in a 24-well plate, with severalwells left empty for blank well staining.

Cells were incubated overnight and had reached confluency at this point.Approximately 1.8 ml of media was removed from each well and replacedwith 2 ml Adipogenesis Induction Media. For negative control wells,expansion medium only was used at this step and for subsequent mediachanges. For all media changes, media was replaced as gently as possibleto avoid disturbing the monolayer.

Cells were incubated for 3 days at 37° C., 5% CO₂, media was replacedwith fresh Adipogenesis Induction Media, and cells were incubated for anadditional three days, at which point adipogenesis could be observed inabout 5-10% of cells. Media was removed and replaced with 2 ml ofmaintenance medium containing either RHAMM antagonist peptides(experimental group) or insulin (concentration as in the inductionmedium used as positive control). Cells were incubated for 3 days at 37°C., 5% CO₂.

Media was removed and BODIPY or Oil Red O staining was performed asdescribed above for assay 1.

Example 2: Identification and Testing of Adipogenic Peptides

We previously showed that ablation of RHAMM expression promotedadipogenesis (Tolg et al., 2006, J Cell Biol). We produced a RHAMMcarboxy-terminal sequence [^(AA681)LDAFEAEKQA LLNEHGATQE QLNKIRDSYAQLLGHQNLKQ KIKHVVKLKD ENSQLKSEVS KLRSQLVKRK QNELRLQGEL DKALGIRHFDPSKAFCHASK ENFTPLKEGN PNCC^(aa794) (SEQ ID NO: 91)] as a recombinantprotein, prepared antibodies to this protein fragment, and showed thatboth the protein fragment and the antibody promoted adipogenesis using acommercially available mesenchymal stem cell kit. The results indicatedthat this 114 amino acid protein fragment contains adipogenic activity.

We hypothesized that the fragment and the antibody were blocking RHAMMfunction by competitive inhibition and by direct binding/blocking ofRHAMM, respectively. Using truncation analyses, we further reduced thesize of the peptide to 32 amino acids (LKQKIKHVVKLKDENSQLKSEVSKLRSQLVKR(SEQ ID NO: 92)) and showed that adipogenic activity is localized to a15 amino acid peptide (KLKDENSQLKSEVSK (SEQ ID NO: 2); “peptide B” or“B-1” herein) shown in FIG. 2. We synthesized this peptide chemically touse as a competitive blocker and we prepared antibodies against thissequence tagged with keyhold lymphocyte hemacyanin (KLH) to directlybind/block RHAMM function. Both reagents effectively block RHAMMfunction and promote adipogenesis in tissue culture and in vivo.

We noted that the RHAMM sequence that contains adipogenic activity alsocontains the hyaluronan binding region of this protein. We thereforeperformed unbiased screening of a random phage library for peptides thatbind to hyaluronan, and identified a hyaluronan binding peptide (SEQ IDNO: 1, peptide P15-1, also referred to as P-1) that similarly promotesadipogenesis in tissue culture and in vivo. The sequence of peptideP15-1 is shown in FIG. 7.

Examples of the adipogenic effects of RHAMM antagonists (peptide B (SEQID NO: 2), peptide P15-1 (SEQ ID NO. 1), and the antibody to peptide B)are summarized in Table 1. Peptide P15-1, peptide B, and a RHAMMantibody were assayed for their effects on pre-adipocyte stem cells inculture and when injected into the dermis of aged rats. Reagents wereranked on a scale of 0-5 with 5 representing the highest possible scoreand a completely filled in Harvey ball. “2D” refers to two-dimensionaltissue culture.

TABLE 1 The adipogenic potential of peptide and antibody reagents. 2Drat Pre-adipocytes (rat In vivo 2D human mesenchymal stem cells) Ratskin Pre-adipocytes peptide P15-1 2 2 2 (SEQ ID NO: 1) peptide B 5 5 5(SEQ ID NO: 2) Antibody 4 4 4

All of these reagents can replace insulin in their ability to promoteadipogenesis in mesenchymal stem cells or papillary fibroblasts.However, peptide B (SEQ ID NO: 2) was the most adipogenic of thesereagents (Table 1) and was therefore further characterized in culturefor dose range activity and several animal models in vivo.

FIG. 3A shows the adipogenic effect of insulin in assays 1 and 2 in bonemarrow mesenchymal stem cells. The negative controls in this experimentwere cells cultured in maintenance medium only.

As shown in FIGS. 3B and 3C, and the micrographs in FIG. 4, in bonemarrow mesenchymal stem cells, a maximum adipogenic effect was obtainedat 1-5 μg of peptide B (SEQ ID NO: 2), depending upon the screeningassay used. FIG. 4 shows the adipogenic effect of peptide B in rat bonemarrow mesenchymal stem cells using assay 1. The arrows in FIG. 4indicate lipid droplets, and “priming stimulus” refers to the treatmentof the cells with dexamethasone and IBMX.

In vivo, 25-50 μg of peptide B (SEQ ID NO: 2)/injection site in a ratdermal skin model of adipogenesis induced significant fat accumulation(fat pads shown in FIG. 10). There was not an obvious difference betweenthe two types of adipogenic reagents in vivo and both were mosteffective in old females (see, FIGS. 5A and 5B). In FIGS. 5A and 5B,data are shown as a ratio of the area (“surface area”) of fat padsexpressed as a ratio of experimental/control values. FIG. 5A shows theeffect of the antibody to peptide B and FIG. 5B shows the effect ofpeptide B.

Peptide B (SEQ ID NO: 2) was also tested for its effect in nude guineapigs since their skin structure resembles human facial skin. Inparticular, the skin of this animal model is more similar to human skinthan is rat skin, in that it is hairless and the keratinocyte layer isthicker than in the rat model. As shown in FIG. 6, H and E stainingrevealed an increase in subcutaneous fat deposition was observed on theupper back of these animals and in the ear fat pad after injection ofpeptide B in both locations (control animals were injected with acollagen gel vehicle only). Thus, peptide B was effective in promotingadipogenesis in this nude guinea pig model.

In order to identify additional adipogenic peptides and to identify thekey adipogenic sequences, two approaches were used. In the first, anunbiased screen was performed to identify hyaluronan-binding peptidesusing 15-mer random phage libraries. Peptide sequences identified inthis screen were sorted for: (a) their alignment to the HA bindingregion of RHAMM, and (b) their lack of alignment to the HA bindingregion of CD44. Peptide P15-1 was identified in this manner. PeptideP15-1 has the sequence STMMSRSHKTRSHHV (SEQ ID NO: 1).

In the second approach, a series of truncations and alanine mutagenesison peptide B (SEQ ID NO: 2) were performed in order to identify thesmallest active sequence. Fragments of peptide B were prepared andassayed for adipogenesis. First, fragments corresponding to the twohalves of peptide B were prepared (KLKDENS (SEQ ID NO:93) and SQLKSEVSK(SEQ ID NO: 44)). Only the SQLKSEVSK (SEQ ID NO: 44) fragment was foundto be adipogenic. This fragment was then further shortened to LKSEVSK(SEQ ID NO: 42) and KSEVSK (SEQ ID NO: 3), both of which were also foundto be adipogenic. Thus, the truncation experiments identified a 9-mer(SQLKSEVSK (SEQ ID NO: 44)), a 7-mer (LKSEVSK (SEQ ID NO: 42)) and a6-mer (KSEVSK (SEQ ID NO: 3)) that exhibited adipogenic activity (seeFIGS. 7, 8, and 9). In FIG. 7, the 7-mer is referred to as Fragment Aand the 9-mer is referred to as Fragment C. In FIGS. 8 and 9, “−vecontrol” refers to the negative control, and “+ve control” refer so thepositive control). FIG. 8 shows the adipogenic effects of the 6-mer(KSEVSK (SEQ ID NO: 3)) in assays 1 and 2. In FIG. 9, the upper panel ofmicrographs shows results from assays 1 and 2, and the lower panel ofmicrographs shows results from assay 1. In the upper panel of FIG. 9,the panels labeled “control” are a positive control (induction plusinsulin). In the lower panel of FIG. 9, the cells in the untreatedcontrol were treated only with maintenance medium.

Peptide P15-1 (SEQ ID NO: 1) was also truncated to create a 9-mer havingthe sequence STMMSRSHK (“Fragment B”; SEQ ID NO: 62). As shown in FIG.7, this 9-mer also has adipogenic activity.

As shown in FIG. 7, these fragments all had approximately the sameadipogenic activity suggesting that the specific activity (activity perweight unit) is highest for the 6-mer KSEVSK (SEQ ID NO: 3). Specificactivities (Table 2) were calculated for peptide B (SEQ ID NO: 2) andthe 9-mer, 7-mer and 6-mer fragments (SEQ ID NOs: 44, 42, and 3,respectively) using adipogenesis data obtained in assays 1 and 2 asdescribed above, and it was unexpectedly found that the 6-mer had thehighest specific activity.

TABLE 2 Specific activity of peptide B and the 9-, 7-, and 6-mers.Specific Molecular Fold adipogenic activity weight induction vs.(activity/ (daltons) negative control 1000 daltons) Assay 1 Peptide B(15mer) 1839 1.85 1.0 (SEQ ID NO: 2) 9mer (SEQ ID NO: 44) 1135 2.4 2.17mer (SEQ ID NO: 42) 884 2 2.3 6mer (SEQ ID NO: 3) 753 2.8 3.7 Assay 215mer (SEQ ID NO: 2) 1839 3.1 1.7 9mer (SEQ ID NO: 44) 1135 3.0 2.6 7mer(SEQ ID NO: 42) 884 3.0 3.4 6mer (SEQ ID NO: 3) 753 4.2 5.6

These results were surprising and unexpected and show that a shorterpeptide is more active than longer peptides. In particular, theincreased specific activity of the 6-mer (KSEVSK; SEQ ID NO: 3) ascompared to peptide B (SEQ ID NO: 2; a 15-mer) was unexpected, sinceboth the 6-mer and peptide B contain the same number key hyaluronanbinding sequences.

The 6-mer (KSEVSK; SEQ ID NO: 3) was tested for adipogenic effect in ratskin in vivo. The results of this testing are shown in the photographsin FIG. 10. The 6-mer is able to induce large fat pads, possibly largerthan peptide B.

To identify the key amino acids needed for an adipogenic effect, alaninemutagenesis of the 6-mer (KSEVSK; SEQ ID NO: 3) was performed. Sincethere appears to be a link between hyaluronan binding and adipogenesisand since hyaluronan binding requires basic residues, the flankinglysine (K) residues were first mutated. As shown in FIGS. 11 and 12, thepeptides ASEVSK (SEQ ID NO: 38) and KSEVSA (SEQ ID NO: 41) both hadreduced adipogenic effects as compared to the KSEVSK (SEQ ID NO: 3)6-mer when tested using assay 1 (FIG. 11) or assay 2 (FIG. 12).

In addition, shorter sequences were also examined and it was discoveredthat pentamers also exhibited adipogenic activity. Pentamers in whicheach of the flanking lysine residues were deleted were also synthesized.As shown in FIG. 13, these pentamers (KSEVS (SEQ ID NO. 82) and SEVSK(SEQ ID NO: 83)) were both much less active than the KSEVSK (SEQ ID NO:3) hexamer, further confirming the importance of the terminal lysineresidues. However, both pentamers still exhibited a low level ofadipogenic activity (in FIG. 13, A2-Ctrl(−)X10 is the negative control,A2-1X10 is a dose of 1 μg, A2-5X10 is a dose of 5 μg, and A2-25X10 is adose of 25 μg; y-axes are absorbance at 520 nm).

The results of truncating the KSEVSK (SEQ ID NO: 3) peptide to pentamerpeptides and of the alanine mutation experiments show that the loss ofthe flanking lysine residues reduces but does not ablate adipogenicactivity, and indicate that presence of the two lysine residues canproduce a peptide that has a high level of adipogenic activity.

To identify a minimal motif sufficient to impart adipogenic activity toa peptide, the KSEVSK (SEQ ID NO: 3) hexamer was used to query the BLASTdatabase to identify evolutionarily conserved sequences in RHAMMproteins from other organisms. The results of this query are shown belowin Table 3.

TABLE 3  Evolutionary conservation of the KSEVSK(SEQ ID NO: 3) sequence in RHAMM proteins from various species.Human, mouse, rat, cow, KSEVSK (SEQ ID NO: 3) and maccaca Goat KSEVLK(SEQ ID NO: 7) Xenopus laevis KQEVSK (SEQ ID NO: 4) Ciona intestinalisKQENTK (SEQ ID NO: 6) Taeniopygia guttata KQDVSK (SEQ ID NO: 8)Danio rerio* KQELDR (SEQ ID NO: 9) KQEVDK (SEQ ID NO: 5) KQEVSK(SEQ ID NO: 4) Thermatoga thermerum LEEIFK (SEQ ID NO: 10)Thermodesulfovibrio LSELEK (SEQ ID NO: 11) yellowstonii *The BLASTdatabase includes Danio rerio RHAMM proteins sequences including each ofthe three listed sequences.

In addition, a BLAST query of the KSEVSK (SEQ ID NO: 3) sequenceidentified a number of similar sequences found in other proteins linkedto both adipogenesis and hyaluronan, for example Sox 2, Sox 21, andupstream binding factor-1. These sequences are shown below in Table 4.

TABLE 4 KSEVSK (SEQ ID NO: 3)-like sequences found in proteins linked toadipogenesis and hyaluronan. Sequence (SEQ ID NO) Source Protein(s)KSEISK (12) cJnk interacting protein 4 KNEVSK (13) Salvador homologue 1KSEVTK (14) interleukin1 receptor accessory protein KSEVNK (15) FAM 111BKSDVSK (16) casp8-associated protein 2 KSQVSK (17)zinc finger protein 432  SEVSK (83)myb binding protein 1A, leucine-rich repeat containingprotein 16B, TOPB1 interacting check point and replication regulatorKSEVS  (82) rho guanine nucleotide exchange factor 5, YLP motifcontaining 1, ASP, mucin 16 KPEVSK (18)proline-rich transmembrane protein 2 KSEVGK (19)cysteine/serine rich nuclear protein 2 KSDSSK (20) nipped B homologueKSSPSK (21) nipped B-like protein KSEASK (22)galanin receptor 1, WD repeating protein, myosine VI KSELRK (23)amyotrophic lateral sclerosis 2 KCEVSK (24) titin isoform novexKSKPSK (25) titin isoform novex KKEVSK (26)actin binding Rho-activation protein, RFX4, regulator ofnonsense transcripts 2 KEEVSK (27) glucocorticoid-induced transcript 1KSETSK (28) ERI1 exoribonuclease family member, tubulin tyrosine ligaseKSNVSK (29) leucine-rich repeat containing protein 8c KDEVSK (30)trasnlocated promoter region, nuclear basket protein KSEVEK (31)hect and RLD domain containing E3 ubiquitin ligase KSAVSK (32)nesprin 1, spectrin repeat containing nuclear envelope 1 KWEVSK (33)renalase, FAD-dependent amine oxidase KMEVSK (34) zinc transporter 5KSEVQK (35) kinectin 1 KSEVHK (36) uncharacterized protein C14orf28KSSVSK (37) upstream binding transcription factor RNA polymerase, zincfinger and BTB domain containing protein 1

The sequences from Tables 3 and 4 were aligned with one another and usedto generate the motifs described herein. In view of the diversity of theamino acids at the second, third, fourth, and fifth positions of thesesequences, it is expected that alanine mutagenesis at these positionswill yield peptides having at least some adipogenic activity. Therefore,in generating the broadest motifs discussed herein, it was assumed thatan alanine residue could be present at the second, third, fourth, orfifth position of the peptide.

In addition, as noted above, peptide P15-1 (SEQ ID NO: 1) was truncatedto create a 9-mer having the sequence STMMSRSHK (“Fragment B”; SEQ IDNO: 62), and this 9-mer was shown to have adipogenic activity.Alignments of peptide P15-1 with the KSEVSK (SEQ ID NO: 3) hexamer usingthe COBALT and MUSCLE alignment tools revealed that the sequence KSEVSKaligns with the STMMSR portion of peptide P15-1, as shown below:

KSEVSK--------- STMMSRSHKTRSHHV

Thus, it is expected that shorter fragments of peptide P15-1 (SEQ ID NO:1), and in particular the hexamer sequence STMMSR (SEQ ID NO: 51), willalso have adipogenic activity. A BLAST query of the STMMSR sequenceidentified a number of similar sequences found in other proteins linkedto both adipogenesis and hyaluronan, for example calcium binding protein1, ribonuclease P protein, ADP ribosylation factor protein 5,transmembrane protein 236, plasma serine protease inhibitor, CD80,lysophosphatidic acid receptor, plasma serine protease inhibitor,angiopoietin 2, ADP-ribosylation factor binding protein, ubiquitinprotein 2, clathrin heavy chain 2, metastasis suppressor protein 1,polycystin 2, and tetratricopeptide repeat protein 17. These sequencesare shown below in Table 5.

TABLE 5  STMMSR-like sequences found in proteinslinked to adipogenesis and hyaluronan. Sequence (SEQ ID NO)Source Protein(s) SIMMSR (52) B-cell/CLL lymphoma protein 9 STLMSR (53)FAM 212B, olfactory receptor 5R1 STVMSR (54)dipeptidyl peptidase 4, splicing factor 3B STGLSR (55)E3 ubiquitin ligase RFWD2 STTMSR (56) metastasis suppressor 1STRMSR (57) E3 ubiquitin ligase, RFWD2 STLMRR (58) mucin 16 STPVSR (59)mucin 16

The sequences in Table 5 were aligned with one another and with theSTMMSR sequence (SEQ ID NO: 51) to generate the motifs described herein.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “comprises”, “including” and “having” are intended to beinclusive and mean that there may be additional elements other than thelisted elements.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods without departingfrom the scope of the invention, it is intended that all mattercontained in the above description and shown in the accompanying figuresshall be interpreted as illustrative and not in a limiting sense. Allpublications, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A pharmaceutical or cosmetic compositioncomprising a pharmaceutically or cosmetically acceptable carrier and amodified peptide or peptidomimetic, wherein the modified peptide orpeptidomimetic is at least 6 amino acids in length, but not more than 8amino acids in length, and comprises a motif having the sequence: KSEVSK(SEQ ID NO: 3), wherein the modified peptide: comprises a protectinggroup, wherein the modified peptide has improved ex vivo and/or in vivopeptide stability and/or improved skin penetration when administeredtopically, relative to an unmodified peptide of the same sequence; or isfunctionalized with a polymer selected from the group consisting of apolyethylene glycol, a cellulose, and a modified cellulose, wherein themodified peptide has increased bioavailability, relative to anunmodified peptide of the same sequence; and wherein the peptidomimeticcomprises one or more peptide linkages replaced with a linkage selectedfrom the group consisting of —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO, wherein the peptidomimetic has animprovement in a property, relative to an unmodified peptide of the samesequence, wherein the property is selected from the group consisting of:economy of production, chemical stability, half-life, absorption,potency, efficacy, and/or reduced antigenicity.
 2. The composition ofclaim 1, wherein the modified peptide or peptidomimetic is 6 amino acidsin length.
 3. The composition of claim 1, wherein the composition is fortopical, subcutaneous, or transdermal administration.
 4. The compositionof claim 1, wherein the composition is for injection.
 5. The compositionof claim 1, wherein the composition further comprises collagen.
 6. Thecomposition of claim 5, wherein the collagen is a bovine collagen, aporcine collagen, or a human collagen.
 7. The composition of claim 5,wherein the collagen is a synthetic collagen.
 8. The composition ofclaim 1, wherein the composition further comprises an anesthetic.
 9. Thecomposition of claim 8, wherein the anesthetic comprises lidocaine. 10.The composition of claim 1, wherein the composition is a skin cream. 11.The composition of claim 1, wherein the modified peptide orpeptidomimetic is a modified peptide comprising a protecting group,wherein the modified peptide has improved ex vivo and/or in vivo peptidestability and/or improved skin penetration when administered topically,relative to an unmodified peptide of the same sequence.
 12. Thecomposition of claim 11, wherein the protecting group comprises anacetyl group at the amino-terminus of the modified peptide orpeptidomimetic.
 13. The composition of claim 1, wherein the modifiedpeptide or peptidomimetic is a modified peptide functionalized with apolymer selected from the group consisting of a polyethylene glycol, acellulose, and a modified cellulose, wherein the modified peptide hasincreased bioavailability, relative to an unmodified peptide of the samesequence.
 14. The composition of claim 1, wherein the modified peptideor peptidomimetic is a peptidomimetic comprising one or more peptidelinkages replaced with a linkage selected from the group consisting of—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—, —COCH₂—, —CH(OH)CH₂—, and —CH₂SO,wherein the peptidomimetic has an improvement in a property, relative toan unmodified peptide of the same sequence, wherein the property isselected from the group consisting of: economy of production, chemicalstability, half-life, absorption, potency, efficacy, and/or reducedantigenicity.
 15. A modified peptide or peptidomimetic of at least 6amino acids in length, but not more than 8 amino acids in length,comprising a motif having the sequence: KSEVSK (SEQ ID NO: 3), whereinthe modified peptide: comprises a protecting group, wherein the modifiedpeptide has improved ex vivo and/or in vivo peptide stability and/orimproved skin penetration when administered topically, relative to anunmodified peptide of the same sequence; or is functionalized with apolymer selected from the group consisting of a polyethylene glycol, acellulose, and a modified cellulose, wherein the modified peptide hasincreased bioavailability, relative to an unmodified peptide of the samesequence; and wherein the peptidomimetic comprises one or more peptidelinkages replaced with a linkage selected from the group consisting of—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—, —COCH₂—, —CH(OH)CH₂—, and —CH₂SO,wherein the peptidomimetic has an improvement in a property, relative toan unmodified peptide of the same sequence, wherein the property isselected from the group consisting of: economy of production, chemicalstability, half-life, absorption, potency, efficacy, and/or reducedantigenicity.
 16. The modified peptide or peptidomimetic of claim 15,wherein the modified peptide or peptidomimetic is 6 amino acids inlength.
 17. The modified peptide or peptidomimetic of claim 15, which isa modified peptide comprising a protecting group, wherein the modifiedpeptide has improved ex vivo and/or in vivo peptide stability and/orimproved skin penetration when administered topically, relative to anunmodified peptide of the same sequence.
 18. The modified peptide orpeptidomimetic of claim 17, wherein the protecting group comprises anacetyl group at the amino-terminus of the peptide or peptidomimetic. 19.The modified peptide or peptidomimetic of claim 15, which is a modifiedpeptide functionalized with a polymer selected from the group consistingof a polyethylene glycol, a cellulose, and a modified cellulose, whereinthe modified peptide has increased bioavailability, relative to anunmodified peptide of the same sequence.
 20. The modified peptide orpeptidomimetic of claim 15, which is a peptidomimetic comprising one ormore peptide linkages replaced with a linkage selected from the groupconsisting of —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—, —COCH₂—, —CH(OH)CH₂—,and —CH₂SO, wherein the peptidomimetic has an improvement in a property,relative to an unmodified peptide of the same sequence, wherein theproperty is selected from the group consisting of: economy ofproduction, chemical stability, half-life, absorption, potency,efficacy, and/or reduced antigenicity.
 21. A method of stimulatingadipogenesis in a subject, the method comprising administering thecomposition of claim 1 to the skin of a subject in an amount sufficientto stimulate adipogenesis.
 22. The method of claim 21, wherein the skincomprises a burn scar, a surgical scar, an acne scar, a biopsy scar, orscarring produced by an injury.
 23. The method of claim 21, wherein thesubject is a human.
 24. A method of stimulating adipogenesis in asubject, the method comprising administering the composition of claim 1subcutaneously to a subject in an amount sufficient to stimulateadipogenesis.
 25. The method of claim 24, wherein the composition isadministered subcutaneously to a lip, an eye lid, a cheek, a forehead, achin, or a neck.
 26. The method of claim 24, wherein the subject ishuman.